Methods and systems for microfluidic screening

ABSTRACT

Provided are methods and systems useful for screening large libraries of effector molecules. Such methods and systems are particularly useful in microfluidic systems and devices. The methods and systems provided herein utilize encoded effectors to screen large libraries of effectors.

CROSS-REFERENCE

This application is a continuation application of U.S. patentapplication Ser. No. 17/553,337 filed Dec. 16, 2021, which is adivisional application of U.S. patent application Ser. No. 17/090,719filed Nov. 5, 2020, now U.S. Pat. No. 11,247,209 issued Feb. 15, 2022,which is a continuation application of U.S. patent application Ser. No.17/067,534 filed Oct. 9, 2020, which claims the benefit of U.S.Provisional Application No. 62/913,624, filed Oct. 10, 2019, and U.S.Provisional Application No. 62/954,348, filed Dec. 27, 2019, each ofwhich is incorporated by reference herein in its entirety.

BACKGROUND

Drug development often requires a significant amount of testing andanalysis to determine how specific chemical substances impact cellularand other biological components. As such, devices and specificmethodologies that focus on correlating a relationship between specificchemical substances and biological components is an integral componentfor a drug developer, such as pharmaceutical companies.

SUMMARY

Provided herein are systems and methods for performing high-throughputassays using microfluidic systems and encoded effectors. The systems andmethods described herein can be used to perform nearly any assay in ahigh-throughput manner and provide detailed information about the effectof various effector molecules on biological systems. The systems andmethods provided herein utilize encoded effectors, which allow a user toreadily ascertain which of the effectors has an effect on a biologicalsample.

Other systems have various drawbacks, including an inability tocustomize the addition of reagents at concentrations of interest atdifferent unit operations during a screen. The systems and methodsdescribed herein address these drawbacks. For example, methods andsystems described herein allow for the introduction of reagents atspecified concentrations at different steps in a screening procedure. Insome instances, adding reagents at defined concentrations allows uniformdoses of effectors to be administered across a library being screened.This may allow for decreased false positives in a screen because lowpotency but highly-loaded effectors may be dosed against samples at auniform concentration across a library screen. In some instances, thecustomizable additions of reagents allow for facile deconvolution ofscreening hits without a step of physical sorting of effectors thatelicit a positive or negative response in the screen.

In another aspect are methods of monitoring biological samples in amicrofluidic based screen without utilizing light (e.g. fluorescence)emitted from a sample. These methods may allow for more detailedinformation about a sample being analyzed than is available by othermethods. Further provided herein are methods and systems forincorporating genetic or cellular information from a sample into theencoded effectors. This incorporation step can allow for an improvedanalysis of the response of a cell or other biological sample contactedwith an effector than is available by other methods. In another aspect,information encoded in a sample, such as a DNA barcode, is incorporatedfrom the sample into the encoding to allow determination of synergisticbenefits of multiple effectors. This can be used for conducting asmall-molecule fragment-based screen to generate compound leads.

The methods provided herein provide advantages over existing DNA encodedlibraries being used for drug screening. In some embodiments, themethods enclosed herein are functional “activity-based” assays, not just“affinity-based” assays: they allows the screening of functional assays.In some embodiments, the methods herein are not limited to testing if acandidate drug binds to a disease target. In contrast, the methodsherein may be capable of testing whether the candidate drug functionsagainst that disease target. Such functions may comprise inhibition,disruption of protein-protein interactions, or activating an enzyme orallosteric pocket.

In some instances, the methods provided herein can screen in complexenvironments such as cell lysates, cells, or other multi-componentmixtures in a single assay. In some embodiments, the functional activitytest is orthogonal to all other components in a mixture and isspecifically testing for functional activity of a target of interest.The screening modalities provided herein are diverse. Such modalitiescan screen for potency, selectivity, toxicity, liabilities, or other keymetrics critical for drug discovery campaigns. The methods providedherein may allow for speed and diversity at 1000 times lower operationalcost than other methods. In some instances, the speed, low reagentneeds, and exceptional validation rates allow fast, iterative screeningof potentially an unlimited set of chemically diverse compounds. Theflexibility and speed allow for testing or screening of compounds inmany different assays or formats for a single target, allowing multiplesampling of conditions, easy “restarts”, fast “hit to lead” starts, and“immediate” validation of library designs.

In some instances, the methods provided herein do not require highsequencing depth, thus reducing costs for analysis. Additionally, themethods disclosed herein may allow for the quantification of yields ofeach chemistry step, allowing normalized dose-response curves andpossibly quantitative analysis.

In some instances, the methods provided herein enable the use of DNAdamaging chemistries that require organic solvents, or conditions thatwould otherwise be DNA damaging in the synthesis of encoded beads. Forexample, some chemistries needed to construct small-molecules maydegrade or cause DNA to become non-amplifiable and thus the DNA barcodeinformation can no longer be read. In some instances, this challenge isovercome by providing DNA encodings bound to scaffolds at high levels.In some embodiments, the scaffolds comprise 10 million or more encodingsbound to a scaffold. Additionally, in some embodiments, as few as 10encodings are required to be present in order to detect a positive hit.

Provided herein are methods for cell phenotypic screening. Cellsdirectly within droplets can be tested and probed for a variety ofdifferent phenotypes. For example, an entire library can be screened fortoxicity against a particular cell type, or an entire library can bescreened for its ability to affect a particular disease target in itsnative cell context, or an entire library can be screened for itsability to affect a panel of targets (transcriptome, protein panel,etc.). This is allowed because a small molecule can be liberated off ofthe bead where it can then penetrate intracellularly a cell (or affectan extracellular target) and affect a particular disease target

Further provided herein are methods for normalizing the results ofscreens of encoded effectors. Other methods of ascertaining the resultsfrom a screen suffer from high rates of false negative results, where aneffector displays potency against a target sample, but due to damage tothe encoding during the screen or low abundance of the encoding duringthe synthesis of the encoded effector, the “hit” is missed in thesubsequent analysis. Provided herein are methods for normalizing theamount of encodings present after a screen has been performed in orderto minimize false negative results due to low abundance of encodings ofpotent effectors.

Also provided herein are devices for performing the methods providedherein. In some instances, the method provided herein are performed onmicrofluidic devices or in microfluidic channels.

Further provided herein are devices useful for the performance ofhigh-throughput screen using encoded libraries. These devices can allowfor fixing a target sample, in some instances a single cell, in a fixedlocation in space with a single encoded effector. Such devices can allowfor screening single compounds against cells to determine desiredeffects without the need to create in situ encapsulations separatingeach individual sample/effector combination.

Disclosed herein, in some embodiments is a method for screening anencoded effector, the method comprising: a) providing at least one celland a scaffold in an encapsulation, wherein the scaffold comprises anencoded effector bound to the scaffold by a photocleavable linker and anucleic acid encoding the effector; b) cleaving the photocleavablelinker to release the encoded effector from the scaffold; and c)detecting a signal from the droplet, wherein the signal results from aninteraction between the encoded effector and the at least one cell. Insome embodiments, cleaving the photocleavable linker releases apre-determined amount of the encoded effector into the droplet. In someembodiments, the photocleavable linker is cleaved using electromagneticradiation. In some embodiments, cleaving the photocleavable linkercomprises exposing the encapsulation to a light from a light source. Insome embodiments, the light intensity of the light is from about 0.01J/cm² to about 200 J/cm². In some embodiments, the method furthercomprising the step of lysing the one or more cells. In someembodiments, the method further comprising providing an activatingreagent to activate the photocleavable linker, so as to enable thephotocleavable linker to be cleaved from the encoded effector.

Disclosed herein, in some embodiments, is a system for screening anencoded effector, the system comprising: a) one or more cells; b) ascaffold, wherein an encoded effector is bound to the scaffold by acleavable linker, wherein a nucleic acid encoding the effector is boundto the scaffold; and c) a microfluidic device configured to: i) receivethe one or more cells and scaffold; ii) encapsulate the one or morecells and scaffold within an encapsulation; iii) cleave the cleavablelinker from the encoded effector to release a predetermined amount ofthe encoded effector within the encapsulation; iv) incubate the encodedeffector with the one or more cells for a period of time; v) detect asignal from the encapsulation, wherein the signal results from aninteraction between the encoded effector and one or more cells; and vi)sort the encapsulation based on the detection of the signal. In someembodiments, the cleavable linker is a photocleavable linker. In someembodiments, the microfluidic device further comprises a firstcollection tube and second collection tube for sorting theencapsulation, wherein the encapsulation is placed in 1) the firstcollection tube if the signal is at or above a predetermined thresholdor 2) the second collection tube if the signal is below a predeterminedthreshold. In some embodiments, the system further comprising a waveformpulse generator to move the encapsulation to the first or secondcollection tube by an electrical field gradient, by sound, by adiaphragm, by modifying geometry of the microfluidic channel, or bychanging the pressure of a microfluidic channel of the microfluidicdevice. In some embodiments, the signal is detected based on detectingmorphological changes in the one or more cells measured by recording aseries of images of the droplet or detecting fluorescence emitted by amolecular beacon or probe. In some embodiments, the period of time iscontrolled by residence time as the encapsulation travels through amicrofluidic channel of the microfluidic device.

Disclosed herein, is a method for amplifying a primer to maximizecellular nucleic acid capture comprising: a) providing an encapsulationcomprising a nucleic acid encoded scaffold with one or more cells, anamplification mix, and a nicking enzyme, wherein a nucleic acid encodingis bound to the nucleic acid encoded scaffold; b) lysing the one or morecells to release one or more cellular nucleic acids; c) nicking thenucleic acid encoding with the nicking enzyme, thereby creating anencoded nucleic acid primer; d) amplifying the encoded nucleic acidprimer via the nicking site and amplification mix; and e) labeling areleased cellular nucleic acid with the encoded nucleic acid primer. Insome embodiments, the specific site comprises a specific nucleotidesequence. In some embodiments, amplifying the encoded nucleic acidprimer comprises 1) creating a copy of the nucleic acid encoding thatextends from the nicking site, and 2) nicking the nucleic acid encodingcopy to create another encoded nucleic acid primer. In some embodiments,amplifying the encoded nucleic acid primer comprises simultaneously 1)creating a copy of the nucleic acid encoding that extends from thenicking site, and 2) displacing the nucleic acid encoding copy to createanother encoded nucleic acid primer. In some embodiments, theamplification mix comprises an amplification enzyme, such that theamplification enzyme enables for a copy of the nucleic acid encoding tobe simultaneously created and displaced. In some embodiments, theamplification enzyme comprises a polymerase. In some embodiments, eachnucleic acid encoding comprises a capture site that prescribes a targetcellular coding or a target cellular nucleic acid to label a releasedcellular nucleic acid.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the disclosure are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present disclosure will be obtained by reference tothe following detailed description that sets forth illustrativeembodiments, in which the principles of the disclosure are utilized, andthe accompanying drawings of which:

FIG. 1 provides a depiction of a nucleic acid encoded effector bound toa bead along with the nucleic acid encoding.

FIG. 2A shows an exemplary workflow of a screen using a nucleic acidencoded effector bound to a bead.

FIG. 2B shows an exemplary workflow of a screen using a microfluidicdevice.

FIG. 2C shows an exemplary workflow for an encapsulation assay screen.

FIG. 2D shows an exemplary workflow for an encapsulation assay screenusing pico-injection.

FIG. 3 illustrates an exemplary method for amplifying a primer tomaximize cellular nucleic acid capture.

FIG. 4 shows an exemplary microfluidic device for performing a screenaccording to the methods provided herein.

FIG. 5 shows an exemplary microfluidic device for performing a screenaccording to the methods provided herein.

FIG. 6 shows an exemplary microfluidic device for performing a screenaccording to the methods provided herein.

FIG. 7 shows an exemplary microfluidic device for performing a screenaccording to the methods provided herein.

FIG. 8 shows an overview of specifically designed IC chips and theirrelated development workflow.

FIG. 9A provides a depiction of an exemplary microfluidic deviceprovided with the methods and systems described herein.

FIG. 9B provides a depiction of a specific section of an exemplarymicrofluidic device provided herein.

FIG. 9C shows a picture of an exemplary microfluidic device providedherein

FIG. 10 provides another exemplary depiction of the microfluidic deviceprovided in FIG. 9A.

FIG. 11 provides a depiction of another exemplary microfluidic deviceused with the methods and systems described herein.

FIG. 12A provides a depiction of a library of beads attached with anencoded-effector modified with fluorophore.

FIG. 12B provides a depiction of an encoded-effector modified with afluorophore dye being liberated from a bead upon being exposed to UVlight.

FIG. 12C provides a depiction of the released encodedeffector-fluorophore from FIG. 12B.

FIG. 12D provides a depiction of the cleavage region of a microfluidicdevice described herein.

FIG. 12E shows a depiction of a correlation between UV light and acalibrant fluid.

FIG. 12F shows a depiction of an exemplary device for confocal laser andPMT emission capture.

FIG. 13A shows measured intensity peaks of a fluorophore dye using 100mV UV light.

FIG. 13B shows a droplet map corresponding to intensity peaks of afluorophore dye using 100 mV UV light.

FIG. 14A shows measured intensity peaks of a fluorophore dye using 600mV UV light.

FIG. 14B shows a droplet map corresponding to intensity peaks of afluorophore dye using 600 mV UV light.

FIG. 15A provides a known correlation between UV power and PMT count ofa fluorophore dye.

FIG. 15B provides a histogram of distributed intensity values of encodedeffector-fluorophore compared with UV power exposure.

FIG. 16 provides exemplary data of UV confinement in a microfluidicdevice described herein.

FIG. 17A-B shows exemplary molecules being activated for photocleavage.

FIG. 18 shows exemplary data for uniform incubation in the microfluidicdevice shown in FIG. 9A

FIG. 19 shows exemplary data for uniform incubation in the microfluidicdevice shown in FIG. 11.

FIG. 20 shows the microfluidic device of FIG. 9A with various detectorpoints along an assay flow path.

FIG. 21 shows an exemplary detection of a specific location along anassay flow path in a microfluidic device described herein.

FIG. 22A-B shows an exemplary fluorescence detection device used with amicrofluidic device described herein, and description of relatedcomponents.

FIG. 23A provides the detection of raw intensity levels at an incubationtime of 0 s for a fluorophore dye.

FIG. 23B provides the detection of real-time smoothing of the intensitylevels from FIG. 23A.

FIG. 24A provides the detection of raw intensity levels at an incubationtime of 1333 s for a fluorophore dye.

FIG. 24B provides the detection of real-time smoothing of the intensitylevels from FIG. 24A.

FIG. 25 shows increasing measured intensity peaks for a fluorophore dyeacross an incubation period of an assay.

FIG. 26A shows an exemplary bead attached with a TR1-TAMRA fluorophore.

FIG. 26B shows an exemplary intensity peak detected for the TR1-TAMRAafter it has been released from the bead.

FIG. 27A shows an exemplary bead attached with a TR3 inhibitor.

FIG. 27B shows an exemplary intensity inhibited corresponding toactivity by the TR3 inhibitor after it has been released from the bead.

FIG. 27C shows an exemplary variation of Cathepsin D activity based onincreasing concentration of a TR3 inhibitor.

FIG. 28A provides an exemplary depiction of a sorting schematic forbeads that exhibit an intensity below an inhibition threshold.

FIG. 28B shows an exemplary intensity peak detected for the TR1-TAMRAthat is above a threshold for positive sorting.

FIG. 28C shows an exemplary intensity peak inhibited for the TR3inhibitor that is below a threshold for positive sorting.

FIG. 28D shows an exemplary a device being used for sortingencapsulations.

DETAILED DESCRIPTION Screening Methods and Systems

Provided herein are methods and systems for screening various effectorsagainst samples in a high-throughput, low-material manner. The systemsand methods, in some embodiments, utilize encoded effectors to probevarious responses from samples. In some embodiments, encoded effectorsare molecules whose structures can be measured by measuring a propertyof the corresponding encoding. Generally, samples are incubated witheffectors in encapsulations. In response to an interaction with theeffector, some type of signal can then be detected. Based on thissignal, the effector can be determined to have efficacy against thesample in inducing a particular response. The systems and methodsdescribed herein, in some embodiments, utilize small encapsulations,such as droplets. In some instances, each individual encapsulationcarries out an assay of the effector and the sample in a small volume. Alarge library of such effectors can be screened against the sample atthe same time and in the same experiment, thus providing high-throughputmethods for conducting screens. Effectors that produce a desired signalfrom a sample can then be sorted, and the encoding of the effector canbe measured to deconvolute which effectors were efficacious in theassay.

Encoded Effectors

The systems and methods provided herein utilize encoded effectors. Anencoded effector, in some embodiments, is an effector that has beenlinked with an encoding such that ascertaining a property of theencoding allows a researcher to readily determine the structure of theeffector. An effector can be any type of molecule or substance whoseeffect on a sample is being investigated. In some embodiments, theeffector is a compound, a protein, a peptide, an enzyme, a nucleic acid,or any other substance. In some instances, the encoding allows a user todetermine the structure of the effector by determining a property of theencoding. Thus, each encoding moiety has a measurable property that,when measured, can be used to determine the structure of the effectorwhich is encoded. Many different encoding modalities can be used,including without limitation nucleic acids and peptides. When theencoding modalities are nucleic acids, the sequence of the nucleic acidmay provide information about the structure of its correspondingeffector. In some instances, the encoded effectors are described by whatkind of molecules is used in the encoding. For example, “nucleic acidencoded effectors” comprise an effector encoded by a nucleic acid.

In some instances, the effectors and their corresponding encodings arebound to a scaffold. This can allow the effector/encoding pair to remainlinked in space. In some instances, when encoded effectors are placedinto solutions or other environments, the link between the pairing isnot lost. Many materials can be used as scaffolds, as any materialcapable of binding both the effector and the encoding may accomplish thedesired goal of keeping the pair linked in space.

Various methods for preparing encoded effectors linked to scaffolds canbe used. In some embodiments, the methods use orthogonal, compatiblemethodologies to create an effector and its encoding in a parallelsynthesis scheme. This is sometimes referred to as “split and poolsynthesis.” For illustrative purposes only, an exemplary, non-limiting,workflow for the preparation of a scaffold containing an effector andencoding is described as follows: A first effector subunit is attachedat an attachment point of a scaffold. The scaffold is then washed toremove unreacted and excess reagents from the scaffold. A first encodingsubunit is then attached at another attachment point on the scaffold,and a wash step performed. Following this, a second effector subunit isthen attached to the first effector subunit, followed by another washstep. Then, a second encoding subunit is attached to the first encodingsubunit, followed by a wash step. This process is repeated as many timesas desired to prepare the desired effectors and corresponding encodings.This process can be repeated on a massively parallel scale in smallvolumes to prepare vast libraries of compounds at low cost and with lowamounts of reagents. In some instances, pre-synthesized compounds areloaded onto scaffolds which contain encodings. The encodings may bepre-synthesized and loaded onto the scaffolds or are synthesizeddirectly onto the scaffolds using methods analogous to the split andpool synthesis described above. In some instances, each scaffoldcomprises numerous copies of a unique effector and its correspondingencoding.

An example of a nucleic acid encoded effector linked with a bead isshown in FIG. 1. A bead linked encoded effector 100 comprises a bead101. Attached at one position is a nucleic acid encoding 102, which iscovalently attached to the scaffold in this example. The nucleic acidencoding comprises encoding subunits A, B, and C. The encoding subunitscorrespond with effector subunits A, B, and C, which make up effector103. The effector 103 is linked to the bead 101 through a linker 104.The linker 104 may be a cleavable linker, such a linker cleavable byelectromagnetic radiation (photocleavable) or selectively cleavable by acleaving reagent (chemically cleavable). Cleavable linkers can be usedto liberate effectors from a bead or other scaffold to allow theeffector to interact with a sample.

In some embodiments, the scaffolds further comprise impurities in theeffector and/or its encoding. In some instances, impurities of theeffector and its corresponding encoding occur due to damage during ascreen, during manufacturing of the bead, effector, or encodingcombination, or during storage. In some embodiments, impurities of theeffector and its corresponding encoding are present due to defects inthe methodologies used to synthesize the encoded effectors. In someembodiments, scaffolds as described herein can comprise a singleencoder, an encoding and its impurities, or combinations thereof. Insome embodiments, at least 5%, at least 10%, at least 20%, at least 30%,at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, or at least 99% of the effectors attached to ascaffold comprise an identical structure. In some embodiments, at least5%, at least 10%, at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, or at least 99% of the encodings attached to a scaffold comprise anidentical structure.

Screening System Components and Methods

Provided herein are methods and systems for screening encoded effectorson samples using encapsulations. In some embodiments, methods andsystems for screening encoded effectors on samples are capable of beingperformed in a high-throughput manner. In some embodiments, the methodsand systems provided herein allow for screening large libraries ofencoded effectors using small volumes, minimal amounts of reagents, andsmall amounts of the effectors being screened. In some embodiments, themethods and systems provided herein allow for uniform dosing ofeffectors in a library against samples. In some embodiments, the methodsand systems described herein allow for measurement of cellular featuresin a high throughput manner. In some embodiments, the methods andsystems provided herein measure genomic, metabolomic, and/or proteomicdata from cells screened against the encoded effectors. In someembodiments, the methods and systems provided herein allow forsynergistic effects of using multiple effectors against a particularsample to be determined. In some embodiments, the methods and systemsprovided herein allow for a library of mutant proteins to be screenedfor a desired activity or improvement in activity.

A non-limiting example workflow of a screen utilizing a single encodedeffector bound to a scaffold is shown in FIG. 2A. The nucleic acidencoded effector bound to a scaffold is encapsulated with a target ofinterest, in this case a cell. In step 1, the effector, in this case adrug, is then cleaved from the bead within the encapsulation. In step 2,the effector is allowed to interact with the cell. If the drug has adesired effect on the cell, a reporter signal indicates that the drug isa positive hit. If there is no reporter signal detected, then the resultfor that drug is negative. In step 3, positive and negative results aresorted based on the detection of the signal. At the end of a screen, instep 4, the positive hits, which have been pooled together, are thensequenced (in the case of nucleic acid encodings) to reveal whicheffectors had the desired effect. In step 5, this information can thenbe used to guide synthesis of further libraries or identify leadmolecules for further development.

FIG. 2B shows an additional exemplary, non-limiting workflow of aneffector screen on a microfluidic device. In the exemplary workflowshown, a nucleic acid encoded effector bound to a bead is placed in aninlet and merged with an additional aqueous stream, which, in someembodiments, contains a sample to be tested. The merged fluids aredriven through an “extrusion region” or “droplet formation region,”wherein beads and sample are encapsulated within a carrier fluidimmiscible with the aqueous fluids. An effector is then cleaved frombead at the effector cleavage region, which in some embodiments utilizesa light source to cleave a photocleavable linker. The encapsulationscontaining cleaved effectors are then allowed to continue flowing alongthe flow path of the device through the incubation region, which in someembodiments contains widened or enlarged chambers to control flow rateor residence time on the device. As the encapsulations travel throughthe incubation region, a detectable signal is generated if the releasedeffectors have a desired activity. This signal is then detected in adetection region of the device. In some embodiments, this detectablesignal is a fluorescent signal, though any detectable signal can beemployed. This signal is then measured or detected at a detectionregion, which is in some embodiments equipped with a light source (e.g.a laser or LED) and a detector (e.g. a photomultiplier tube (PMT), acharged coupled device (CCD), or a photodiode) coupled to a sortingdevice (e.g. a dielectrophoresis electrode or any other sortingmechanism). In some embodiments, the detection region comprises aninterrogation region, which is coupled to a sensor or an array ofsensors. Based on the signal, the encapsulations are sorted into a wasteoutlet or a hit outlet. Following completion of the screen, theencodings of the hits are amplified (e.g. by PCR or emulsion PCR) andthe encodings sequenced (e.g. by next generation sequencing). Thesequenced encodings can then be decoded to reveal the effectors whichhad the desired activity. In some embodiments, each bead furthercomprises barcode unique to the bead itself (independent of theeffector). Thus, in some embodiments, it is possible to ascertain ifmultiple beads bearing identical effectors were selected as hits withinmultiple encapsulations.

An exemplary, non-limiting droplet assay screen workflow is shown inFIG. 2C. A bead buffer comprising a probe substrate and nucleic acidencoded effectors bound to beads are merged with an assay buffercomprising a disease target (e.g. a protein such as an enzyme). Anencapsulation comprising probe substrate, a bead bearing a nucleic acidencoded effector, and the disease target is then formed in an immisciblecarrier fluid. The effector is then released from the bead and allowedto interact with the disease target. The sample is then incubated withina delay line (or any such suitable channel or reservoir configured toincubate the encapsulation for a desired time). In this embodiment, theprobe substrate is cleaved by the disease target. Upon cleavage, achange in fluorescence properties of the substrate is observed, forexample due to FRET interactions of the probe substrate. If the diseasetarget is inhibited by the effector, the probe substrate will not becleaved. After a desired incubation time, the fluorescence of theencapsulation is measured (e.g. by a PMT, CCD, or photodiode) afterexcitation (e.g. by a laser or LED) and the encapsulation is sorted(e.g. by electrophoresis or dielectrophoresis) based on the result. FIG.2D shows a similar workflow but contains an additional step of adding asubstrate detection reagent (e.g. by pico-injection or droplet merging)in order to allow detection of substrate that has or has not reactedwith the disease target. In some embodiments, an electrode is employedat the pico-injection site in order to destabilize the interface of theencapsulation to facilitate incorporation of the pico-injected fluidinto the encapsulation.

Provided herein are methods and systems for screening encoded effectorson samples using encapsulations, wherein the sample and an encodedeffector are encapsulated. In some embodiments, the encoded effector andthe sample are encapsulated by mixing a first solution comprising theencoded effector with a second solution comprising the sample. In someembodiments, the first and second solutions are mixed together with anoil. In some embodiments, mixing the first and second solutions with anoil forms an emulsion, wherein the first and second solutions combine toform droplets. In some embodiments, encapsulations are formed in amicrofluidic device. In some embodiments, the encapsulation stepcomprises merging the first and second solution at a T-junction ofmicrofluidic channels. In some embodiments, creating an encapsulationcomprises converging aqueous streams in a microfluidic device. Creatingan encapsulation can occur by numerous methods, any of which may becompatible with the methods described herein. In some embodiments,encapsulations are formed on microfluidic devices. In some embodiments,encapsulations flow through a microfluidic device.

In some embodiments, provided herein are methods and systems forscreening a library of encoded effectors. In some embodiments, for anymethod or system described herein, the library of encoded effectorscomprises at least about 1, 1,000, 10,000, 100,000, 250,000, 1,000,000,or 10,000,000 unique encoded effectors. In some embodiments, a pluralityof scaffolds (as described herein) are encapsulated in a plurality ofencapsulations (as described herein) with a sample in a microfluidicchannel. In some embodiments, the plurality of scaffolds (e.g., beads)are bound to a library of unique encoded effectors. In some embodiments,each scaffold is bound to one or more unique encoded effectors. In someembodiments, the library of unique encoded effectors comprise at leastabout 250,000 unique encoded effectors. In some embodiments, the libraryof unique encoded effectors comprise about 1 unique encoded effector toabout 10,000,000 unique encoded effectors. In some embodiments, thelibrary of unique encoded effectors comprise about 1 unique encodedeffector to about 1,000 unique encoded effectors, about 1 unique encodedeffector to about 10,000 unique encoded effectors, about 1 uniqueencoded effector to about 100,000 unique encoded effectors, about 1unique encoded effector to about 250,000 unique encoded effectors, about1 unique encoded effector to about 1,000,000 unique encoded effectors,about 1 unique encoded effector to about 10,000,000 unique encodedeffectors, about 1 unique encoded effector to about 200 unique encodedeffectors, about 1,000 unique encoded effectors to about 10,000 uniqueencoded effectors, about 1,000 unique encoded effectors to about 100,000unique encoded effectors, about 1,000 unique encoded effectors to about250,000 unique encoded effectors, about 1,000 unique encoded effectorsto about 1,000,000 unique encoded effectors, about 1,000 unique encodedeffectors to about 10,000,000 unique encoded effectors, about 1,000unique encoded effectors to about 200 unique encoded effectors, about10,000 unique encoded effectors to about 100,000 unique encodedeffectors, about 10,000 unique encoded effectors to about 250,000 uniqueencoded effectors, about 10,000 unique encoded effectors to about1,000,000 unique encoded effectors, about 10,000 unique encodedeffectors to about 10,000,000 unique encoded effectors, about 10,000unique encoded effectors to about 200 unique encoded effectors, about100,000 unique encoded effectors to about 250,000 unique encodedeffectors, about 100,000 unique encoded effectors to about 1,000,000unique encoded effectors, about 100,000 unique encoded effectors toabout 10,000,000 unique encoded effectors, about 100,000 unique encodedeffectors to about 200 unique encoded effectors, about 250,000 uniqueencoded effectors to about 1,000,000 unique encoded effectors, about250,000 unique encoded effectors to about 10,000,000 unique encodedeffectors, about 250,000 unique encoded effectors to about 200 uniqueencoded effectors, about 1,000,000 unique encoded effectors to about10,000,000 unique encoded effectors, about 1,000,000 unique encodedeffectors to about 200 unique encoded effectors, or about 10,000,000unique encoded effectors to about 200 unique encoded effectors,including increments therein. In some embodiments, the library of uniqueencoded effectors comprise about 1 unique encoded effector, about 1,000unique encoded effectors, about 10,000 unique encoded effectors, about100,000 unique encoded effectors, about 250,000 unique encodedeffectors, about 1,000,000 unique encoded effectors, about 10,000,000unique encoded effectors, or about 200 unique encoded effectors. In someembodiments, the library of unique encoded effectors comprise at leastabout 1 unique encoded effector, about 1,000 unique encoded effectors,about 10,000 unique encoded effectors, about 100,000 unique encodedeffectors, about 250,000 unique encoded effectors, about 1,000,000unique encoded effectors, or about 10,000,000 unique encoded effectors.In some embodiments, the library of unique encoded effectors comprise atmost about 1,000 unique encoded effectors, about 10,000 unique encodedeffectors, about 100,000 unique encoded effectors, about 250,000 uniqueencoded effectors, about 1,000,000 unique encoded effectors, about10,000,000 unique encoded effectors, or about 200 unique encodedeffectors.

In some embodiments, each unique encoded effector is encoded with acorresponding encoding. In some embodiments, at least one encodingcomprises a nucleic acid encoding. In some embodiments, at least oneencoded effector is bound to a respective scaffold through a cleavablelinker. In some embodiments, the cleavable linker comprises aphotocleavable linker, or a chemically cleavable linker (e.g., linkercleaved through contact with a reagent). In some embodiments, one ormore photocleavable linkers between an encoded effector andcorresponding bead is cleaved. In some embodiments, cleaving aphotocleavable linker releases the corresponding encoded effector fromthe bead. In some embodiments, a released encoded effector interactswith the corresponding sample within the respective encapsulation. Insome embodiments, the interaction between the encoded effector and thesample creates a signal. In some embodiments, the signal is configuredto be detected. In some embodiments, the plurality of encapsulations aresorted based on a corresponding signal being detected from eachencapsulation. In some embodiments, the plurality of encapsulations aresorted based on a corresponding signal not being detected from eachencapsulation. In some embodiments, the encoding(s) associated with theencapsulations having a detected signal(s) are barcoded, as analternative sorting the encapsulation. In some embodiments, theencoding(s) associated with the encapsulations not having a detectedsignal(s) are barcoded, as an alternative sorting the encapsulation. Insome embodiments, encapsulations are formed on microfluidic devices. Insome embodiments, encapsulations flow through a microfluidic device.

Provided herein are methods and systems for screening encoded effectorson samples using encapsulations, wherein a signal is detected from theencapsulation. In some embodiments, the signal results from aninteraction between an effector and the sample. In some embodiments, thesignal is detected with a detector. In some embodiments, detecting thesignal comprises providing the encapsulation through a microfluidicchannel. In some embodiments, detecting the signal comprises providingthe encapsulation through a microfluidic channel equipped with adetector. In some embodiments, the detector is configured to detect thesignal.

Signals of the methods and systems provided herein can be any signalcapable of detection in an encapsulation. In some embodiments, thesignal is electromagnetic radiation, thermal radiation, a visual changein the sample, or combinations thereof. In some embodiments, theelectromagnetic radiation is fluorescence or luminescence. In someembodiments, the electromagnetic radiation is in the visible spectrum.In some embodiments, the signal is absorbance of electromagneticradiation.

Provided herein are methods and systems for screening encoded effectorson samples using encapsulations, wherein the encapsulation is sorted. Insome embodiments, the encapsulation is sorted based on the detection ofa signal. In some embodiments, the encapsulation is optionally sortedbased on the detection of a signal.

Alternative Signal Detection

Provided herein are methods and systems for screening encoded effectors,wherein various alternative signal detection methods and systems may beused to identify activity by an effector or within an encapsulation. Insome embodiments, the signal is a thermal radiation. In someembodiments, the thermal radiation is detected using an infrared camera.In some embodiments, the thermal radiation is a change in thermalradiation emitted by a sample. In some embodiments, the change inthermal radiation is due to metabolic activity in a sample. In someembodiments, the change in thermal radiation comprises a change inmetabolic activity in the sample. In some embodiments, the change inthermal radiation comprises a change in metabolic activity in the sampledue to an effect of the effector on the sample. In some embodiments theeffect on the sample is a change in metabolic activity. In someembodiments, detecting the signal comprises detecting a change inmetabolic activity in the sample by detecting a change in thermalradiation. In some embodiments, the sample is a cell and the signal isthermal radiation.

In some embodiments, the sample displays a change in emission of thermalradiation compared to a sample not encapsulated with the effector. Insome embodiments, the change in thermal radiation is at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% emission of thermalradiation. In some embodiments, the change in thermal radiation is atleast 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% emission ofthermal radiation relative to sample not treated with the effector. Insome embodiments, the change in thermal radiation is at least 2-fold,3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-foldemission of thermal radiation relative to a sample not treated with theeffector.

In some embodiments, the signal is luminescence. In some embodiments,detecting the signal comprises monitoring encapsulations for a period oftime. In some embodiments, detecting the signal comprises monitoringluminescence from the sample over a period of time. In some embodiments,the luminescence is integrated over a period of time. In someembodiments, the luminescence is integrated over a period of at least 1minute, at least 5 minutes, at least 30 minutes, at least 4 hours, or atleast 12 hours. In some embodiments, the luminescence is integrated overa period of at most 1 minutes, at most 5 minutes, at most 30 minutes, atmost 4 hours, or at most 12 hours. In some embodiments, the luminescenceis integrated over a distance traveled by an encapsulation. In someembodiments, the luminescence is integrated over a distance travelled byan encapsulation through a microfluidic channel. In some embodiments,the luminescence is integrated over a distance of at least 1 μm, atleast 10 μm, at least 50 μm, at least 100 μm, at least 250 μm, at least500 μm, at least 1 mm, at least 10 mm, or at least 100 mm travelled byan encapsulation through a microfluidic channel. In some embodiments,the luminescence is integrated over a distance of at most 1 μm, at most10 μm, at most 50 μm, at most 100 μm, at most 250 μm, at most 500 μm, atmost 1 mm, at most 10 mm, or at most 100 mm travelled by anencapsulation through a microfluidic channel.

The signal from the sample may be a morphological or visual change inthe sample which can be measured by imaging the encapsulation. In someembodiments, detecting the signal comprises recording images of thesample in the encapsulation. In some embodiments, detecting the signalcomprises recording a series of images of the sample in theencapsulation. In some embodiments, detecting a signal comprisesrecording a series of images of samples in encapsulations andsuperimposing the series of images of the sample. In some embodiments,detecting a signal comprises detecting morphological or visual changesin the sample measured by recording a series of images of theencapsulation.

In some embodiments, morphology changes in a sample, such as one or morecells, can be detected by an imaging sensor, capturing trans illuminatedlight with a high-speed shutter, where composite video frames offersmultiple full-cell images that can aid in shape determination. In someembodiments, morphology changes in a sample, such as one or more cells,can be detected by an imaging sensor, capturing trans illuminated lightfrom a high-frequency pulsed light source, increasing temporalresolution and sharpening the perimeter of the cell. In onemanifestation, morphology changes can be detected by fluorescenceemission from a cell traversing a laser-light sheet excitation region.In some embodiments, the emission is captured by Avalanche Photodiode(APD) or charged coupled detector (CCD), in a one-dimensional array ofpixels, binned by time, then restitched into a compositefluorescence-microscopy image.

In some embodiments, detecting the signal comprises recording images ofthe sample, wherein the sample is a cell. In some embodiments, recordingimages of the cell provides information about cell morphology, mitoticstage, levels of expressed proteins, levels of cellular components, cellhealth, or combinations thereof. In some embodiments, the encapsulationcomprises a detection agent. In some embodiments, the detection agent isan intercalation dye. In some embodiments, the intercalation dye isethidium bromide, propidium iodide, crystal violet, a dUTP-conjugatedprobe, DAPI (4′,6-diamidino-2-phenylindole), 7-AAD (7-aminoactinomycinD), Hoechst 33258, Hoechst 33342, Hoechst 34580, combinations thereof,or derivatives thereof In some embodiments, the detection agenthighlights different regions of the cell. In some embodiments, thedetection agent highlights a particular organelle. In some embodiments,the organelle is a mitochondrion, Golgi apparatus, endoplasmicreticulum, nucleus, ribosomes, cellular membrane, nucleolus, liposome,lipid vesicle, lysosome, or vacuole. In some embodiments, the organelleis a mitochondrion. In some embodiments, the organelle is the nucleus.

In some embodiments, detecting the signal comprises detecting thepresence of a target nucleic acid. In some embodiments, theencapsulation further comprises a molecular beacon. In some embodiments,the molecular beacon is complementary to a portion of the target nucleicacid sequence of the sample. In some embodiments, the methods furthercomprise adding a molecular beacon to the encapsulation. In someembodiments, the target nucleic acid is detected by a molecular beacon.In some embodiments, the encapsulation further comprises a probe and apolymerase. In some embodiments, the encapsulation further comprises aTaqMan probe and a Taq polymerase. In some embodiments, the methodsfurther comprise adding a TaqMan probe and a Taq polymerase to theencapsulation. In some embodiments, the TaqMan probe is complementary toa portion of the target nucleic acid sequence. In some embodiments, theTaqMan probe and Taq polymerase are added to the encapsulation at thesame time. In some embodiments, the TaqMan probe and Taq polymerase areadded sequentially. In some embodiments, the signal is fluorescenceemitted by a molecular beacon. In some embodiments, the signal isfluorescence emitted by TaqMan probe. In some embodiments, the signal isfluorescence emitted by a molecular beacon or TaqMan probe.

Various molecular beacons can be used with the methods and systemsdescribed herein. In general, a molecular beacon comprises a nucleicacid binding region that binds to a complementary nucleic acid ofinterest. The molecular beacon can typically have a secondary structurewherein a fluorophore and a quencher are in proximity when the nucleicacid binding region is not bound to the complementary nucleic acid ofinterest. Upon binding of the nucleic acid binding region to thecomplementary nucleic acid of interest, the fluorophore and quencher maybe separated in space such that a fluorescent signal can be detected.Thus, the amount of fluorescence detected can be used to quantify theamount of nucleic acid of interest present in a sample. In someembodiments, an inhibitor is used wherein activity between an effectorand a sample inhibits or limits the intensity of a fluorescence signal.

In some embodiments, two or more signal detection methods are used incombination for detecting a signal. In some embodiments, detecting asignal comprises detecting morphological changes in the sample as wellas detecting fluorescence emitted by a molecular beacon or probe. Forexample, in some embodiments, fluorescence emission from a molecularbeacon in the encapsulation (e.g., droplet) can be measured by PMT orAvalanche Photodiode (APD). In some embodiments, simultaneous imagecapture by transillumination can identify other features in theencapsulation (e.g., droplet), such as encoded effectors and cells. Insome embodiments, these streams of information together determineoutcome at the sorting junction.

In some embodiments, detecting the presence of the target nucleic acidcomprises amplifying the target nucleic acid. In some embodiments, thetarget nucleic acid is amplified by an isothermal amplification method.In some embodiments, the isothermal amplification method isloop-mediated isothermal amplification (LAMP), strand displacementamplification (SDA), helicase-dependent amplification (HAD), recombinasepolymerase amplification (RPA), rolling circle replication (RCA) ornicking enzyme amplification reaction (NEAR). In some embodiments, theencapsulation further comprises reagents for isothermal amplification ofthe target nucleic acid. In some embodiments, the methods compriseadding reagents for isothermal amplification to the encapsulation. Insome embodiments, the reagents for isothermal amplification are specificto the target nucleic acid sequence.

In some embodiments, the target nucleic acid is DNA. In someembodiments, the target nucleic acids are cellular DNA. In someembodiments, the target nucleic acids are genomic DNA. In someembodiments, the target nucleic acid is RNA. In some embodiments, theRNA is mRNA, ribosomal RNA, tRNA, non-protein-coding RNA (npcRNA),non-messenger RNA, functional RNA (fRNA), long non-coding RNA (lncRNA),pre-mRNAs, or primary miRNAs (pri-miRNAs). In some embodiments, thetarget nucleic acids are mRNA.

Scaffold and Beads

An exemplary embodiment of screening encoded effectors on samples usingencapsulations comprises use of a scaffold. In some embodiments, theeffector is bound to a scaffold. In some embodiments, the scaffold actsas a solid support and keeps the encoded effector molecules linked inspace to their encodings. In some embodiments, the scaffold is astructure with a plurality of attachment points that allow linkage ofone or more molecules. In some embodiments, the encoded effector isbound to a scaffold. In some embodiments, the scaffold is a solidsupport. In some embodiments, the scaffold is a bead, a fiber,nanofibrous scaffold, a molecular cage, a dendrimer, or a multi-valentmolecular assembly.

In some embodiments, the scaffold is a bead. In some embodiments, thebead is a polymer bead, a glass bead, a metal bead, or a magnetic bead.In some embodiments, the bead is a polymer bead. In some embodiments,the bead is a glass bead. In some embodiments, the bead is a metal bead.In some embodiments, the bead is a magnetic bead.

The beads utilized in the methods provided herein may be made of anymaterial. In some embodiments, the bead is a polymer bead. In someembodiments, the bead comprises a polystyrene core. In some embodiments,the beads are derivatized with polyethylene glycol. In some embodiments,the beads are grafted with polyethylene glycol. In some embodiments, thepolyethylene glycol contains reactive groups for the attachment of otherfunctionalities, such as effectors or encodings. In some embodiments,the reactive group is an amino or carboxylate group. In someembodiments, the reactive group is at the terminal end of thepolyethylene glycol chain. In some embodiments, the bead is a TentaGel®bead.

The polyethylene glycol (PEG) attached to the beads may be any size. Insome embodiments, the PEG is up to 20 kDa. In some embodiments, the PEGis up to 5 kDa. In some embodiments, the PEG is about 3 kDa. In someembodiments, the PEG is about 2 to 3 kDa.

In some embodiments, the PEG group is attached to the bead by an alkyllinkage. In some embodiments, the PEG group is attached to a polystyrenebead by an alkyl linkage. In some embodiments, the bead is a TentaGel® Mresin.

In some embodiments, the bead comprises a PEG attached to a bead throughan alkyl linkage and the bead comprises two bifunctional species. Insome embodiments, the beads comprise surface modification on the outersurface of the beads that are orthogonally protected to reactive sitesin the internal section of the beads. In some embodiments the beadscomprise both cleavable and non-cleavable ligands. In some embodiments,the bead is a TentaGel® B resin.

Beads for use in the systems and methods as described herein can be anysize. In some embodiments, the beads are at most 10 nm, at most 100 nm,at most 1 μm, at most 10 μm, or at most 100 μm in diameter. In someembodiments, the beads are at least 10 nm, at least 100 nm, at least 1μm, at least 10 μm, or at least 100 μm in diameter. In some embodiments,the beads are about 10 μm to about 100 μm in diameter.

In some embodiments, the effector is covalently bound to the scaffold.In some embodiments, the effector is non-covalently bound to thescaffold. In some embodiments, the effector is bound to the scaffoldthrough ionic interactions. In some embodiments, the effector is boundto the scaffold through hydrophobic interactions.

Cleavable Linker and Effector Release

Cleavable linkers can be used to attach effectors to scaffolds. In someembodiments, the effector is bound to a scaffold by a cleavable linker.In some embodiments, the cleavable linker is cleavable byelectromagnetic radiation, an enzyme, a chemical reagent, heat, pHadjustment, sound, or electrochemical reactivity. In some embodiments,the cleavable linker is cleavable by electromagnetic radiation. In someembodiments, the cleavable linker is cleavable by electromagneticradiation such as UV light. In some embodiments, the cleavable linker isa photocleavable linker. In some embodiments the photocleavable linkeris cleavable by electromagnetic radiation. In some embodiments thephotocleavable linker is cleavable through exposure to light. In someembodiments, the light comprises UV light. In some embodiments, thecleavable linker is cleavable by a cleaving reagent. In someembodiments, the cleavable linker must first be activated in order to beable to be cleaved. In some embodiments, the cleavable linker isactivated through interaction with a reagent.

In some embodiments, the cleavable linker is a disulfide bond. In someembodiments, the cleavable linker is a disulfide bond and the cleavablereagent is a reducing agent. In some embodiments, the reducing agent isa disulfide reducing agent. In some embodiments, the disulfide reducingagent is a phosphine. In some embodiments, the reducing agent is2-mercapto ethanol, 2-mercaptoethylamine, tris(2-carboxyethyl)phosphine(TCEP), dithiothreitol, a combination thereof, or a derivative thereof.

In some embodiments, the cleavable linker and cleaving reagent arebiorthogonal reagents. Bioorthogonal reagents are combinations ofreagents that selectively react with each other, but do not havesignificant reactivity with other biological components. Such reagentsallow for minimal cross-reactivity with other components of the reactionmixture, which allows for less off target events.

In some embodiments, the cleavable linker is a substitutedtrans-cyclooctene. In some embodiments, the cleavable linker is asubstituted trans-cyclooctene and the cleaving reagent is a tetrazine.In some embodiments, the cleavable linker as the structure

wherein X is —C(═O)NR—, —C(═O)O—, —C(═O)— or a bond, and R is H oralkyl. In some embodiments, the cleaving reagent is a tetrazine. In someembodiments, the cleaving reagent is dimethyl tetrazine (DMT). Furtherexamples of tetrazine cleavable linkers and methods of use are describedin Tetrazine-triggered release of carboxylic-acid-containing moleculesfor activation of an anti-inflammatory drug, Chem Bio Chem 2019, 20,1541-1546, which is hereby incorporated by reference.

In some embodiments, the cleavable linker comprises an azido groupattached to the same carbon as an ether linkage. In some embodiments,the cleavable linker has the structure

In some embodiments, the cleaving reagent is a reagent that reduces anazido group. In some embodiments, the cleaving reagent is a phosphine.In some embodiments, the cleaving reagent is hydrogen and a palladiumcatalyst.

In some embodiments, the cleavable linker is cleaved by a transitionmetal catalyst. In some embodiments, the cleavage reagent is atransition metal catalyst. In some embodiments, the transition metalcatalyst is a ruthenium metal complex. In some embodiments, thecleavable linker is an O-allylic alkene. In some embodiments, thecleavable linker has the structure

A non-limiting example of such a catalyst is described in Bioorthogonalcatalysis: a general method to evaluate metal-catalyzed reaction in realtime in living systems using a cellular luciferase reporter system,Bioconjugate Chem. 2016, 27, 376-382, which is hereby incorporated byreference. In some embodiments, the transition metal complex is apalladium complex. In some embodiments, the cleavable linker has thestructure

Such cleavable linkers are described in 3′-O-modified nucleotides asreversible terminators for pyrosequencing, PNAS Oct. 16, 2007, 104 (42)16462-16467, which is hereby incorporated by reference.

In some embodiments, the number of effectors cleaved from the scaffoldis controlled. In some embodiments, the number of effectors cleaved froma scaffold is controlled by controlling the amount of stimulus used tocleave the cleavable linker. In this context, a “stimulus” is any methodor chemical used to specifically cleave a cleavable linker. In someembodiments, the stimulus is a chemical reaction with a cleavingreagent. In some embodiments, the stimulus is electromagnetic radiation.In some embodiments, the stimulus is a change in pH. In someembodiments, the change in pH is acidification. In some embodiments, thechange in pH is basification.

In some embodiments, methods described herein comprise cleaving thecleavable linker with a cleaving reagent. In some embodiments, themethods comprise adding the cleaving reagent to an encapsulationcomprising an effector bound to a scaffold through a cleavable linker.In some embodiments, the methods comprise adding the cleaving reagent toan encapsulation comprising an encoding bound to a scaffold through acleavable linker.

In some embodiments, the number of effectors cleaved from the scaffoldis controlled by controlling the concentration of the cleaving reagent.In some embodiments, the concentration of the cleavage reagent iscontrolled in an encapsulation containing an encoded effector bound to ascaffold. In some embodiments, the concentration of chemical reagentused to cleave the cleavable linker is at least 100 pM, at least 500 pM,at least 1 nM, at last 10 nM, at least 100 nM, at least 1 μM, at least10 μM, at least 100 μM, at least 1 mM. at least 10 mM, at least 100 mM,or at least 500 mM. In some embodiments, the concentration of cleavingreagent used to cleave the cleavable linker is at most 100 pM, at most500 pM, at most 1 nM, at most 10 nM, at most 100 nM, at most 1 μM, atmost 10 μM, at most 100 μM, at most 1 mM, at most 10 mM, at most 100 mM,or at most 500 mM.

In some embodiments, the cleaving reagent is added to a plurality ofencapsulations. In some embodiments, the concentration of cleavingreagent added to the plurality of encapsulations is substantiallyuniform among individual encapsulations of the plurality. In someembodiments, the concentration of cleaving reagent used to cleave thecleavable linker in a plurality of encapsulations is at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, or 90% identical in each individualencapsulation. In some embodiments, concentration of cleaving reagentused to cleave the cleavable linker in a plurality of encapsulationsdiffers by no more than 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 50-fold, or 100-fold amongeach individual encapsulation of the plurality.

In some embodiments, the cleaving reagent is added to the encapsulationby pico-injection. In some embodiments, the encapsulation is passedthrough a microfluidic channel comprising a pico-injection site. In someembodiments, pico-injections are timed such that the rate ofpico-injection matches the rate at which encapsulation cross thepico-injection site. In some embodiments, at least 80%, 85%, 90%, 95%,98%, or 99% of encapsulations passing a pico-injection site receive apico-injection. In some embodiments, the pico-injections are at least2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least30-fold, at least 50-fold, at least 100-fold, at least 500-fold, or atleast 1000-fold smaller in volume than the passing droplets. In someembodiments, the cleaving reagent is added to the encapsulation bydroplet merging.

In some embodiments, the cleaving reagent is added from a stock solutionto the encapsulation. In some embodiments, the stock solution is atleast 2×, 5×, 10×, 20×, 30×, 50×, 100×, 500×, or 1000× more concentratedthan the desired final concentration in the encapsulation.

In some embodiments, methods and systems described herein comprisecleaving a photocleavable linker between an encoded effector and ascaffold. In some embodiments, the methods and systems described hereincomprise exposing an encapsulation to electromagnetic radiationcomprising an effector bound to a scaffold through a photocleavablelinker. In some embodiments, the methods and systems described hereincomprise exposing an encapsulation to light (for e.g., UV light)comprising an effector bound to a scaffold through a photocleavablelinker. In some embodiments, the encapsulation is exposed to the lightusing a microfluidic device.

In some embodiments, the photocleavable linker is cleaved by exposure tolight (e.g., UV light). In some embodiments, the concentration of thenumber of effector molecules released from a scaffold is controlled bycontrolling the intensity and/or duration of exposure to UV light. Insome embodiments, the light intensity of a light (e.g., UV light) thatan encapsulation (e.g., droplet) described herein is exposed to is atleast about 0.1 J/cm² to about 200 J/cm². In some embodiments, the lightintensity of a light (e.g., UV light) that an encapsulation (e.g.,droplet) described herein is exposed to is about 0.1 J/cm² to about 200J/cm². In some embodiments, the light intensity of a light (e.g., UVlight) that an encapsulation (e.g., droplet) described herein is exposedto is about 0.1 J/cm² to about 5 J/cm², about 0.1 J/cm² to about 25J/cm², about 0.1 J/cm² to about 100 J/cm², about 0.1 J/cm² to about 150J/cm², about 0.1 J/cm² to about 200 J/cm², about 5 J/cm² to about 25J/cm², about 5 J/cm² to about 100 J/cm², about 5 J/cm² to about 150J/cm², about 5 J/cm² to about 200 J/cm², about 25 J/cm² to about 100J/cm², about 25 J/cm² to about 150 J/cm², about 25 J/cm² to about 200J/cm², about 100 J/cm² to about 150 J/cm², about 100 J/cm² to about 200J/cm², or about 150 J/cm² to about 200 J/cm², including incrementstherein. In some embodiments, the light intensity of a light (e.g., UVlight) that an encapsulation (e.g., droplet) described herein is exposedto is about 0.1 J/cm², about 5 J/cm², about 25 J/cm², about 100 J/cm²,about 150 J/cm², or about 200 J/cm². In some embodiments, the lightintensity of a light (e.g., UV light) that an encapsulation (e.g.,droplet) described herein is exposed to is at least about 0.1 J/cm²,about 5 J/cm², about 25 J/cm², about 100 J/cm², or about 150 J/cm². Insome embodiments, the light intensity of a light (e.g., UV light) thatan encapsulation (e.g., droplet) described herein is exposed to is atmost about 5 J/cm², about 25 J/cm², about 100 J/cm², about 150 J/cm², orabout 200 J/cm².

In some embodiments, the light (e.g., UV light) that an encapsulation(e.g., droplet) described herein is exposed to is at least about 5 mV.In some embodiments, the light (e.g., UV light) that an encapsulation(e.g., droplet) described herein is exposed to is from about 5 mV toabout 10,000 mV. In some embodiments, the light (e.g., UV light) that anencapsulation (e.g., droplet) described herein is exposed to is about100 mV, 200 mV, 400 mV, 600 mV, 800 mV, 1000 my, 1250 mV, 1500 mV, 2000mV, 4000 mV, 5000 mV. In some embodiments, the light that anencapsulation (e.g., droplet) is exposed to is a calibrated amount oflight.

In some embodiments, the cleavable linker is cleaved by electromagneticradiation. In some embodiments, the concentration of the number ofeffector molecules released from a scaffold is controlled by controllingthe intensity or duration of electromagnetic radiation.

Any suitable photoreactive or photocleavable linker can be used as acleavable linker cleaved by electromagnetic radiation (e.g., exposure toUV light). A non-limiting list of linkers cleavable by electromagneticradiation includes (i) o-nitrobenzyloxy linkers, (ii) o-nitrobenzylaminolinkers, (iii) α-substituted o-nitrobenzyl linkers, (iv) o-nitroveratryllinkers, (v) phenacyl linkers, (vi) p-alkoxyphenacyl linkers, (vii)benzoin linkers, (viii) pivaloyl linkers, and (ix) other photolabilelinkers. Further examples of photocleavable linkers are described inPhotolabile linkers for solid-phase synthesis, ACS Comb Sci. 2018 Jul.9; 20(7):377-99, which is hereby incorporated by reference. In someembodiments, the cleavable linker is an o-nitrobenzyloxy linker, ano-nitrobenzylamino linker, an α-substituted o-nitrobenzyl linker, ano-nitroveratryl linker, a phenacyl linker, p-alkoxyphenacyl linker, abenzoin linker, or a pivaloyl linker.

In some embodiments, the photocleavable linker requires to be firstactivated through exposure to a reagent before being able to be cleavedthrough exposure to electromagnetic radiation (e.g., UV light). In someembodiments, the desired number of effectors released can be furthercontrolled by selectively exposing reagents to encapsulations (e.g.,droplets). In some embodiments, providing photocleavable linkers thatneed to be activated before being cleaved through exposure to UV lightenables for improved bead-handling, synthesis, storage, and preparationdue to minimized or eliminated encoded effector release through incidentUV exposure. FIG. 17A provides an exemplary molecule configured to betransformed upon interaction with a reagent, such that it becomesactivated for UV photocleavage (reference: J. AM. CHEM. SOC. 2003, 125,8118-8119; 10.1021/ja035616d). As depicted, the azide group functionallyreduces the sensitivity of the photocleavable-linker moiety, such thatlinker is more stable, thus advantageous for handling and storing underambient lighting. As depicted in FIG. 17A, the azide can be convertedupon reagent treatment (HOF—CH3CN) to generate the photo-sensitiveNitro-benzyl motif (molecule depicted in the middle), wherein theproduct photocleavable-linker can be calibrated to release a knownquantity of effector upon UV-exposure. FIG. 17B provides anotherexemplary molecule configured to be transformed upon interaction with areagent, such that it becomes activated for UV photocleavage (reference:J. Comb. Chem. 2000, 2, 3, 266-275). As depicted, the thio-phenol esterprovides a stable covalent linker to compound (R). Specific oxidation ofthe thio-phenol (shown in middle molecule) can generate an “activated”linker-moiety. Kinetic control of the oxidation step may allow forquantitative “activation” to prescribe compound release. In someembodiments, base treatment causes linker scission through elimination,thereby generating a free acid compound, or with subsequentdecarboxylation generates just a compound.

In some embodiments, the cleavable linker is cleaved by an enzyme. Insome embodiments, the cleavable linker is cleaved by a protease, anuclease, or a hydrolase. In some embodiments, the cleavable linker is apeptide. In some embodiments, the cleavable linker is a cleavablenucleic acid sequence. In some embodiments, the cleavable linker is acarbohydrate. In some embodiments, the number of effector moleculescleaved from the scaffold is controlled by controlling the concentrationof the enzyme. In some embodiments, the rate at which effector moleculesare cleaved from the scaffold is controlled by controlling theconcentration of the enzyme.

In some embodiments, the methods comprise cleaving the cleavable linker.In some embodiments, the methods comprise cleaving the cleavable linkerwith a cleaving reagent. In some embodiments, the cleaving reagent isadded to the encapsulation by pico-injection. In some embodiments, thecleaving reagent is added to the encapsulation by pico-injection at aconcentration configured to release a predetermined amount of effector.In some embodiments, the cleaving reagent is added to the encapsulationby pico-injection at a concentration configured to release a desiredamount of effector.

In some embodiments, methods described herein comprise first activatingthe cleavable linker to enable the cleavable linker to be cleaved. Insome embodiments, upon activating the cleavable linker, the cleavablelinker can be cleaved using methods described herein, such as throughphotocleavage, interaction with an enzyme, using a cleaving reagent, andso on. In some embodiments, the cleavable linker is activated throughinteraction with an activating reagent. In some embodiments, the methodscomprise adding the activating reagent to an encapsulation comprising aneffector bound to a scaffold. In some embodiments, the methods compriseadding the activating reagent to an encapsulation comprising an encodingbound to a scaffold. In some embodiments, the activating reagentcomprises any reagent described herein as a cleaving reagent. In someembodiments, the activating reagent comprises a disulfide reducingreagent. In some embodiments, the activating reagent comprisestetrazine.

In some embodiments, the activating reagent is added to theencapsulation by pico-injection. In some embodiments, the encapsulationis passed through a microfluidic channel comprising a pico-injectionsite. In some embodiments, pico-injections are timed such that the rateof pico-injection matches the rate at which encapsulation cross thepico-injection site. In some embodiments, at least 80%, 85%, 90%, 95%,98%, or 99% of encapsulations passing a pico-injection site receive apico-injection. In some embodiments, the pico-injections are at least2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least30-fold, at least 50-fold, at least 100-fold, at least 500-fold, or atleast 1000-fold smaller in volume than the passing droplets. In someembodiments, the activating reagent is added to the encapsulation bydroplet merging.

In some embodiments, the concentration of the activating reagent used toactivate the cleavable linker is at most 100 picomolar (pM), at most 500pM, at most 1 nanomolar (nM), at most 10 nM, at most 100 nM, at most 1micromolar (□M), at most 10 □M, at most 100 □M, at most 1 millimolar(mM), at most 10 mM, at most 100 mM, or at most 500 mM.

In some embodiments, the activating reagent is added from a stocksolution to the encapsulation. In some embodiments, the stock solutionis at least 2×, 5×, 10×, 20×, 30×, 50×, 100×, 500×, or 1000× moreconcentrated than the desired final concentration in the encapsulation.

In some embodiments, effectors are released from scaffolds. In someembodiments, releasing effectors from scaffolds allows the effectors tomove freely in solution. This free movement may allow the effector tointeract with the sample or target being interrogated. In someembodiments, these effectors are released in a controlled fashion. Thiscontrolled fashion may allow for a predetermined and/or known dose ofeffectors to be released form the scaffold. Such a procedure may allowfor improved quantification and analysis of hits from a screen, as doseresponse can be measured. Additionally, releasing a known amount ofeffectors across a library of effectors being screened may remove biasfrom the sample set. Bias can occur in library screens using encodedscaffolds when individual scaffolds possess attachments of effectorsthat vary in amount among the scaffolds of the library. For example, onescaffold may contain 10 copies of an effector molecule, and anotherscaffold may contain 1000 copies of an effector molecule. Consequently,different concentrations of effector being screened against a sample ortarget may be released, making a determination of the efficacy ofindividual effectors difficult to ascertain. By releasing a uniformamount of effectors from each scaffold in a screen, a uniform doseacross the screen is employed, removing bias from lower potency, higherconcentration effectors.

In some embodiments, the effectors are released to a desiredconcentration. In some embodiments, the effectors are released to adesired concentration within an encapsulation. In some embodiments, thedesired concentration is at least 100 pM, at least 500 pM, at least 1nM, at least 10 nM, at least 100 nM, at least 1 μM, at least 10 μM, atleast 100 μM, at least 1 mM. at least 10 mM, at least 50 mM, at least100 mM, or at least 250 mM. In some embodiments, the desiredconcentration is at most 100 pM, at most 500 pM, at most 1 nM, at most10 nM, at most 100 nM, at most 1 μM, at most 10 μM, at most 100 μM, atmost 1 mM, at most 10 mM, at most 50 mM, at most 100 mM, or at most 250mM.

In some embodiments, the effectors are released to a predeterminedconcentration. In some embodiments, the effectors are released to apredetermined concentration within an encapsulation. In someembodiments, the predetermined concentration is at least 100 pM, atleast 500 pM, at least 1 nM, at least 10 nM, at least 100 nM, at least 1μM, at least 10 μM, at least 100 μM, at least 1 mM. at least 10 mM, atleast 50 mM, at least 100 mM, or at least 250 mM. In some embodiments,the predetermined concentration is at most 100 pM, at most 500 pM, atmost 1 nM, at most 10 nM, at most 100 nM, at most 1 μM, at most 10 μM,at most 100 μM, at most 1 mM, at most 10 mM, at most 50 mM, at most 100mM, or at most 250 mM.

In some embodiments, effector molecules are released from scaffolds in aplurality of encapsulations. In some embodiments, the concentration ofeffector molecules released from scaffolds in a plurality ofencapsulations is uniform among the encapsulations. In some embodiments,the concentration of effector molecules released from scaffolds in aplurality of encapsulations is substantially uniform among theencapsulations. In some embodiments, the concentration of effectormolecules released from scaffolds in a plurality of encapsulations is atleast 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% identical in eachindividual encapsulation. In some embodiments, the concentration ofeffector molecules released from scaffolds in a plurality ofencapsulations differs by no more than 2-fold, 3-fold, 4-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 50-fold, or100-fold among each individual encapsulation of the plurality.

In some embodiments, the methods described herein comprise incubatingthe encapsulation for a period of time. In some embodiments, the methodscomprise incubating the encapsulation for a period of time to allow theeffector and sample to interact. In some embodiments, the encapsulationsare incubated for a period of time to allow the effector and the sampleto react. In some embodiments, the period of time is at least 1millisecond, 1 second, 1 minute, at least 10 minutes, at least 1 hour,at least 4 hours, or at least 24 hours. In some embodiments, the periodof time is at most 1 minutes, at most 10 minutes, at most 1 hour, atmost for hours, or at most 24 hours. In some embodiments, the incubationtime is measured after releasing effectors from a scaffold.

In some embodiments, the period of time is controlled by a residencetime as the encapsulation travels through a microfluidic channel. Insome embodiments, the residence time is controlled by a flow valve, ageometry of the microfluidic channel, the length of the microfluidicchannel, by removing the encapsulations from the microfluidic channel,or combinations thereof.

The effectors of the methods and systems provided herein can be any typeof molecule. In some embodiments, an effector is a biochemical,chemical, or biological moiety. In some embodiments, an effector is acell, a protein, peptide, small molecule, small molecule fragment, or anucleic acid. An effector is any molecule that is capable interactingwith a target. The term “effector” is used broadly to encompass anymoiety whose effect on a sample is being interrogated.

In some embodiments, the effectors have a handle that allows forattachment to a scaffold. A handle is a reactive functional group thatcan be used to tether the effector to an attachment site on a scaffold.This handle may be any functional group capable of forming a bond.Handles may include, without limitation, sulfhydryl groups, CLICKchemistry reagents, amino groups, carboxylate groups, or numerous othergroups.

In some embodiments, effectors are comprised of individual subunits.These individual subunits may be joined using various chemical reactionsto form the full effector. In some embodiments, iterative chemicalprocesses are used to generate the effectors, similar to methodologiesused in solid-phase peptide synthesis. Similar methods can be used tocreate non-peptide effectors, wherein a first reaction is performed tolink two subunits, the two linked subunits are subjected to a secondreaction to activate the linked subunits, and a third subunit is thenattached, and so on. Any type of such an iterative chemical synthesisscheme may be employed to create the effectors used in the methods andsystems provided herein.

In some embodiments, the effectors elicit a response from the targetbeing interrogated. The response elicited can take any form and dependson the sample being interrogated. As a non-limiting example, when thesample comprises a cell, the response may be a change in expressionpattern, apoptosis, expression of a particular molecule, or amorphological change in the cell. As another non-limiting example, whenthe sample comprises a protein, the effector may inhibit proteinactivity, enhance protein activity, alter protein folding, or measureprotein activity.

In some embodiments, the effector is a protein. In some embodiments, theprotein may be a naturally occurring or mutant protein. In someembodiments, the protein is a fragment of a naturally occurring protein.In some embodiments, the protein is an antibody. In some embodiments,the protein is an antibody-fragment. In some embodiments, the protein isan enzyme. In some embodiments, the protein is a recombinant protein. Insome embodiments, the protein is a signaling protein, an enzyme, abinding protein, an antibody or antibody fragment, a structural protein,a storage protein, or a transport protein, or any mutant thereof

In some embodiments, the effector is a peptide. In some embodiments, theeffector is a non-natural peptide. In some embodiments, the effector isa polymer. In some embodiments, the peptide is 5 amino acids to 50 aminoacids in length. In some embodiments, the peptide is 5 amino acids to 10amino acids, 5 amino acids to 15 amino acids, 5 amino acids to 20 aminoacids, 5 amino acids to 30 amino acids, 5 amino acids to 50 amino acids,10 amino acids to 15 amino acids, 10 amino acids to 20 amino acids, 10amino acids to 30 amino acids, 10 amino acids to 50 amino acids, 15amino acids to 20 amino acids, 15 amino acids to 30 amino acids, 15amino acids to 50 amino acids, 20 amino acids to 30 amino acids, 20amino acids to 50 amino acids, or 30 amino acids to 50 amino acids inlength. In some embodiments, the peptide is 5 amino acids, 10 aminoacids, 15 amino acids, 20 amino acids, 30 amino acids, or 50 amino acidsin length. In some embodiments, the peptide comprises at least 5 aminoacids, 10 amino acids, 15 amino acids, 20 amino acids, or 30 aminoacids. In some embodiments, the peptide comprises at most 10 aminoacids, 15 amino acids, 20 amino acids, 30 amino acids, or 50 aminoacids. In some embodiments, the peptide comprises unnatural amino acids.In some embodiments, the peptide comprises a non-peptide region. In someembodiments, the peptide is a cyclic peptide. In some embodiments, thepeptide has a secondary structure that mimics a protein.

In some embodiments, the effector is a compound. In some embodiments,the compound is an organic molecule. In some embodiments, the compoundis an inorganic molecule. In some embodiments, the compounds used aseffectors contain organic and inorganic atoms. In some embodiments, thecompound is a drug-like small molecule. In some embodiments, thecompound is an organic compound. In some embodiments, the compoundcomprises one or more inorganic atoms, such as one or more metal atoms.In some embodiments, the effector is a small molecule. In someembodiments, the effector is a macro molecule.

In some embodiments, the compound is a completed chemical that issynthesized by connecting a plurality of chemical monomers to eachother. In some embodiments, the effector is a pre-synthesized compoundloaded onto a bead after synthesis.

In some embodiments, the compound is a small molecule fragment. Smallmolecule fragments are small organic molecules which are small in sizeand low in molecular weight. In some embodiments, the small moleculefragments are less than 500 Dalton (Da), less than 400 Da, less than 300Da, less than 200 Da, or less than 100 Da in molecular weight (MW).

In some embodiments, the effector is an effector nucleic acid. In someembodiments, the effector nucleic acid is 5 nucleotides to 50nucleotides in length. In some embodiments, the effector nucleic acid is5 nucleotides to 10 nucleotides, 5 nucleotides to 15 nucleotides, 5nucleotides to 20 nucleotides, 5 nucleotides to 30 nucleotides, 5nucleotides to 50 nucleotides, 10 nucleotides to 15 nucleotides, 10nucleotides to 20 nucleotides, 10 nucleotides to 30 nucleotides, 10nucleotides to 50 nucleotides, 15 nucleotides to 20 nucleotides, 15nucleotides to 30 nucleotides, 15 nucleotides to 50 nucleotides, 20nucleotides to 30 nucleotides, 20 nucleotides to 50 nucleotides, or 30nucleotides to 50 nucleotides in length. In some embodiments, theeffector nucleic acid comprises 5 nucleotides, 10 nucleotides, 15nucleotides, 20 nucleotides, 30 nucleotides, or 50 nucleotides. In someembodiments, the effector nucleic acid comprises at least 5 nucleotides,10 nucleotides, 15 nucleotides, 20 nucleotides, or 30 nucleotides. Insome embodiments, the effector nucleic acid is at most 10 nucleotides,15 nucleotides, 20 nucleotides, 30 nucleotides, or 50 nucleotides inlength. In some embodiments, the effector nucleic acid comprisesunnatural nucleotides. In some embodiments, the nucleic acid is anaptamer. In some embodiments, the effector nucleic acid comprises DNA,RNA, or combinations thereof.

Enzyme Evolution Screen

The methods and systems herein are further useful for screening effectorproteins for the possession of various activities. In these embodiments,the effector is a protein. A variety of mutant variants of a protein canbe screened by linking plasmids or other nucleic acids coding for theexpression of a protein to scaffolds. The “coding” referred to in thisaspect refers to the genetic code, and “encoding” refers to analternative strategy for elucidating the structure of the protein. Insome instances, each nucleic acid has a barcode that is unique to thespecific mutant protein which can be sequenced to reveal the mutationstherein without conducting a full sequence read on the whole plasmid orother nucleic acid which codes for the protein. The barcode thus acts asits own encoding to delineate the structure and sequence of the proteinwithout relying on the full coding sequence. In this aspect, a libraryof mutant proteins can be screened against samples with the componentsprovided herein in encapsulation-based assays.

In a non-limiting example, a scaffold containing the nucleic acidencoding the protein of interest is encapsulated. The protein can thenbe expressed in the encapsulation using an expression system, such asany in vitro transcription/translation system. In some embodiments, oneor more detection reagents can be added to the encapsulation for whichthe protein may exhibit a certain desired activity. In some instances,these detection reagents may be present during the expression of theprotein or may be added later. These detection reagents may be used toassess any desired activity, including protein binding, enzymaticactivity, or the detection reagents may be capable of probing proteinstructure. In some embodiments, each detection reagent comprises one ormore chemical compounds or molecules, which the expressed protein (e.g.,an enzyme of interest) can bind together. In some embodiments, at leasttwo detection reagents are provided, each comprising a molecular probe,such that the expressed protein (e.g., an enzyme) can bind the molecularprobes from the respective detection reagents together. In someembodiments, at least two detection reagents are provided, eachcomprising one or more chemical compounds, such that the expressedprotein (e.g., an enzyme) can bind one or more of the chemical compoundsfrom the respective detection reagents together. In some embodiments,the binding of the molecular probes or chemical compounds by the proteinleads to the production of a signal. In some embodiments, the binding ofthe molecular probes or chemical compounds by the protein is a certaindesired activity that leads to the production of a signal.

If the protein in an encapsulation has the certain desired activity, theactivity can lead to the production of a signal. The signal can be anyof the signals described herein. In some embodiments, the signal is afluorescent signal created due to the ligation of two molecules ofinterest. In some embodiments, the molecules of interest have FRETpairings affixed to them, or fluorophore/quencher pairings affixed tothem, or any other type of moieties that lead to a change in signal dueto brining the two moieties into proximity to each other. In someembodiments, the two moieties are brought into proximity to each otherdue to the formation of a bond between the molecules of interest. Thesignal produced can then be detected, indicating that the protein beingscreened has the desired activity. The encapsulation can then be sortedbased on the detectable signal, such as the signals presence, absence,or level. In some embodiments, the encoded effector is a protein and theencoding comprises a barcoded nucleic acid which further codes for theexpression of the protein.

Provided herein are methods for screening nucleic acid encoded proteinsagainst a sample. In some embodiments, the methods comprise providing anencapsulation comprising a nucleic acid encoding attached to a scaffold,the nucleic acid encoding comprising an encoding barcode and a codingsection for the expression of an encoded effector protein. In someembodiments, the encapsulation further comprises an expression systemfor the production of the encoded protein. In some embodiments, theencoded protein is expressed within the encapsulation. In someembodiments, detection reagents are introduced to the encapsulation. Insome embodiments the detection reagents are present in the encapsulationduring protein expression. In some embodiments, the detection reagentsproduce a signal upon interaction with the encoded protein if theencoded protein has a certain activity. In some embodiments, the signalproduced due to this interaction is measured. In some embodiments, theencapsulation is sorted based on the measurement of the signal. In someembodiments, the nucleic acid encoding is sequenced. In someembodiments, this nucleic acid encoding is sequenced by next-generationsequencing.

The nucleic acid encoding which comprises a coding section for theexpression of the encoded protein may be of any form that allows for theexpression to occur. In some embodiments, the nucleic acid encodingcomprising a coding section for the expression of an encoded effectorprotein is a linear nucleic acid. In some embodiments, the nucleic acidencoding comprising a coding section for the expression of an encodedeffector protein is a plasmid. In some embodiments, the nucleic acidencoding comprising a coding section for the expression of an encodedeffector protein is single stranded. In some embodiments, the nucleicacid encoding comprising a coding section for the expression of anencoded effector protein is double stranded.

In some embodiments, the nucleic acid encoding comprising a codingsection for the expression of an encoded effector protein comprises abarcode. In some embodiments, the barcode acts as the encoding for theencoded effector protein. In some embodiments, the barcode is upstreamof the coding section for the expression of the encoded effectorprotein. In some embodiments, the barcode is downstream of the codingsection for the expression of the encoded effector protein. In someembodiments, the nucleic acid encoding comprising a coding section forthe expression of an encoded effector protein further comprises asequencing primer. In some embodiments, the sequencing primer isupstream of the barcode. In some embodiments, the sequencing primer isdownstream of the barcode.

In some embodiments, the effector is a nucleic acid encoded protein. Insome embodiments, the corresponding nucleic acid encoding comprises acoding section for the expression of the encoded protein. In someembodiments, the nucleic acid encoded protein is an enzyme or mutantthereof. In some embodiments, the enzyme or mutant thereof is beingscreened for an enzymatic activity.

In some embodiments, the enzymatic activity is oxidation, reduction,ligation, polymerization, bond cleavage, bond formation, orisomerization. In some embodiments, the enzymatic activity is covalentbond formation. In some embodiments, the enzyme is an amino aciddehydrogenase, a natural amine dehydrogenase, an opine dehydrogenase, oran imine reductase. In some embodiments, the enzymatic activity is anenantiospecific activity. In some embodiments, the enzymatic activity isa stereospecific activity.

A variety of protein characteristics can be probed or screened for usingthe methods and systems provided herein. In some embodiments, thecertain characteristic being screened for comprises an enzymaticactivity, a binding ability, a catalytic activity, a physical property,an inhibitory activity, or a structure. In some embodiments, the certaincharacteristic being screened for comprises a binding ability. In someembodiments, the certain characteristic being screened for comprises acatalytic activity. In some embodiments, the certain characteristicbeing screened for comprises a physical property. In some embodiments,the certain characteristic being screened for comprises an inhibitoryactivity. In some embodiments, the certain characteristic being screenedfor comprises a secondary, tertiary, or quaternary structure.

In some embodiments, the enzymatic activity is the ability to form abond between molecular probes from a first detection reagent and asecond detection reagent. In some embodiments, the enzymatic activitycomprises forming a bond between molecular probes from a first detectionreagent and a second detection reagent. In some embodiments, theenzymatic activity is the ability to form a bond between one or morechemical compounds from a first detection reagent and a second detectionreagent. In some embodiments, the enzymatic activity comprises forming abond between one or more chemical compounds from a first detectionreagent and a second detection reagent. In some embodiments, the bond isa covalent bond. In some embodiments, the bond is an irreversiblecovalent bond. In some embodiments, the first detection reagent and thesecond detection reagent exhibit a fluorescent signal when the moleculesfrom the first and second detection reagents are bound together. In someembodiments, the first detection reagent and the second detectionreagent exhibit a changed fluorescent signal when molecular probes fromthe first and second detection reagents are bound together compared towhen the molecular probes from the first detection reagent and seconddetection reagent are not bound together. In some embodiments, the firstdetection reagent and the second detection reagent exhibit a fluorescentsignal when the one or more chemical compounds from the first and seconddetection reagents are bound together. In some embodiments, the firstdetection reagent and the second detection reagent exhibit a changedfluorescent signal when one or more chemical compounds from the firstand second detection reagents are bound together compared to when theone or more chemical compounds from the first detection reagent andsecond detection reagent are not bound together. In some embodiments,the fluorescent signal is due to fluorescence resonance energy transfer(FRET), bioluminescent resonance energy transfer (BRET), lanthanidechelate excite time resolved fluorescence resonance energy transfer(LANCE TR-FRET), or an amplified luminescent proximity homogeneousassay. In some embodiments, the first and second reagents are chemicalcompounds.

In some embodiments, the molecular probes from the first and seconddetection reagents comprise a FRET pair or a fluorophore/quencher pair.In some embodiments, the molecular probes from the first and seconddetection reagents comprise fluorophores or quenchers independentlyselected from 4-(4-dimethylaminophenyl azo),5-((3-aminoethyl)amino)-1-napthalene sulfonic acid,5-((2-aminoethyl)amino)-1-napthalene sulfonic acid (EDANS),4-(dimethylaminoazo)benzene-4-carboxylic acid (DABCYL), andfluorescein-isothiocyanate (FITC), or derivatives thereof. In someembodiments, the FRET pair or fluorophore/quencher pair comprisedifferent fluorophores. In some embodiments, the FRET pairing isduplicate copies of the same fluorophore.

In some embodiments, the one or more chemical compounds from the firstand second detection reagents comprise a FRET pair or afluorophore/quencher pair. In some embodiments, the one or more chemicalcompounds from the first and second detection reagents comprisefluorophores or quenchers independently selected from4-(4-dimethylaminophenyl azo), 5-((3-aminoethyl)amino)-1-napthalenesulfonic acid, 5-((2-aminoethyl)amino)-1-napthalene sulfonic acid(EDANS), 4-(dimethylaminoazo)benzene-4-carboxylic acid (DABCYL), andfluorescein-isothiocyanate (FITC), or derivatives thereof. In someembodiments, the FRET pair or fluorophore/quencher pair comprisedifferent fluorophores. In some embodiments, the FRET pairing isduplicate copies of the same fluorophore.

In some embodiments, the ability to form a bond is an imine reduction.In some embodiments, the imine reduction is enantiospecific. In someembodiments, the imine reduction is stereospecific. In some embodiments,the imine reduction favors an S-enantiomer at a substituted carbonadjacent to the reduced imine bond. In some embodiments, the iminereduction favors an R-enantiomer at a substituted carbon adjacent to thereduced imine bond. In some embodiments, the imine reduction is anintramolecular reaction. In some embodiments, the imine reduction isdiastereospecific.

In some embodiments, a library of nucleic acid encoded proteins arescreened against the sample. In some embodiments, the methods compriseperforming any of the described screens against a library of nucleicacid encoded proteins, wherein the library of nucleic acid encodedproteins comprises a plurality of different mutant versions of thenucleic acid encoded protein. In some embodiments, each mutant versionof the nucleic acid encoded protein is encoded by a unique barcode.

The methods and systems provided herein sometimes comprise the additionof detection reagents to the encapsulation. In some embodiments, thedetection reagents are added by pico-injection. In some embodiments, thedetection reagents are added by droplet merging. In some embodiments,the detection reagents are added before the signal is detected. In someembodiments, the detection reagents are added after the signal isdetected. In some embodiments, the detection reagents facilitate thedetection of the signal.

In some embodiments, the encapsulation further comprises a reporterenzyme. In some embodiments, the reporter enzyme reacts with anotherreagent to produce a functional readout. In some embodiments, a bondbetween the first and second molecular probes creates a new moleculethat inhibits the reporter enzyme.

Additional reagents may also be used to add barcodes to nucleic acids ofthe sample or the encoding. In some embodiments, the additional reagentsadd a nucleic acid barcode to one or more contents of the encapsulation.In some embodiments, the nucleic acid barcode is added to the encoding.In some embodiments, the nucleic acid barcode is added to nucleic acidsfrom the sample.

Encodings for Effectors

The effectors provided herein can be linked with encodings. In someembodiments, the effectors are linked with an encoding. In someinstances, the encoding allows a user to determine the structure of theeffector by determining a property of the encoding. Thus, each encodingmoiety has a measurable property that, when measured, can be used todetermine the structure of the effector which is encoded.

In some embodiments, the encoding is a nucleic acid. In someembodiments, the sequence of the nucleic acid provides information aboutthe structure of the effector. In some embodiments, the encodingcomprises a nucleic acid barcode. In some embodiments, the barcode isunique to a specific effector. In some embodiments, the encodingcomprises a sequencing primer. In some embodiments, sequencing thenucleic acid encoding allows the user to ascertain the structure of thecorresponding effector.

In some embodiments, the encoding is DNA. In some embodiments, theencoding is double stranded DNA. In some embodiments, the encoding issingle stranded DNA. In some embodiments, the encoding is RNA. In someembodiments, the encoding is single stranded RNA. In some embodiments,the encoding is double stranded RNA.

In some embodiments, the encoding nucleic acid comprises at least 20nucleotides, at least 40 nucleotides, at least 60 nucleotides, at least80 nucleotides, at least 100 nucleotides, at least 200 nucleotides, orat least 500 nucleotides. In some embodiments, the encoding nucleic acidcomprises 20 nucleotides to 100 nucleotides in length. In someembodiments, the encoding nucleic acid is 20 nucleotides to 40nucleotides, 20 nucleotides to 60 nucleotides, 20 nucleotides to 80nucleotides, 20 nucleotides to 100 nucleotides, 40 nucleotides to 60nucleotides, 40 nucleotides to 80 nucleotides, 40 nucleotides to 100nucleotides, 60 nucleotides to 80 nucleotides, 60 nucleotides to 100nucleotides, or 80 nucleotides to 100 nucleotides in length. In someembodiments, the encoding nucleic acid comprises about 20 nucleotides,about 40 nucleotides, about 60 nucleotides, about 80 nucleotides, orabout 100 nucleotides. In some embodiments, the encoding nucleic acidcomprises at least 20 nucleotides, 40 nucleotides, 60 nucleotides, or 80nucleotides. In some embodiments, the encoding nucleic acid is at most40 nucleotides, 60 nucleotides, 80 nucleotides, or 100 nucleotides inlength.

In some embodiments, the encoding is made up of individual subunits thatencode a corresponding effector subunit. Consequently, an entireencoding can specify which individual subunits have been linked orcombined to form the effector. In some embodiments, each subunit maycomprise up to 5, 10, 15, 20, 25, 30, 40, 50, or more individualnucleotides. The full encoding sequence can comprise any number of theseindividual subunits. In some embodiments, the full encoding sequencecomprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more encoding subunits.These encoding subunits can be ligated together using many knownmethods, including enzymatic ligation, template-free synthesis,templated polymerase extension, chemical ligation, recombination, orsolid phase nucleic acid synthesis techniques.

In some embodiments, the encoding is a molecular weight barcode. In someembodiments, the molecular weight barcode is at least 1,000, at least5,000, at least 10,000, or at least 15,000 Daltons in molecular weight.In some embodiments, the molecular weight barcode is a peptide. In someembodiments, the molecular weight barcode peptide comprises 5 aminoacids to 10 amino acids, 5 amino acids to 15 amino acids, 5 amino acidsto 20 amino acids, 5 amino acids to 30 amino acids, 5 amino acids to 50amino acids, 10 amino acids to 15 amino acids, 10 amino acids to 20amino acids, 10 amino acids to 30 amino acids, 10 amino acids to 50amino acids, 15 amino acids to 20 amino acids, 15 amino acids to 30amino acids, 15 amino acids to 50 amino acids, 20 amino acids to 30amino acids, 20 amino acids to 50 amino acids, or 30 amino acids to 50amino acids. In some embodiments, the molecular weight barcode peptidecomprises 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids,30 amino acids, or 50 amino acids. In some embodiments, the molecularweight barcode peptide comprises at least 5 amino acids, 10 amino acids,15 amino acids, 20 amino acids, or 30 amino acids. In some embodiments,the peptide comprises at most 10 amino acids, 15 amino acids, 20 aminoacids, 30 amino acids, or 50 amino acids. In some embodiments, themolecular weight barcode peptides comprise unnatural amino acids.

In some embodiments, the encoding is loaded onto a scaffold. In someembodiments, the scaffold comprises a high loading of the encoding. Insome embodiments, the scaffold comprises about 1,000,000 copies to about50,000,000 copies of the encoding. In some embodiments, the scaffoldcomprises about 1,000,000 copies to about 2,000,000 copies, about1,000,000 copies to about 5,000,000 copies, about 1,000,000 copies toabout 10,000,000 copies, about 1,000,000 copies to about 15,000,000copies, about 1,000,000 copies to about 20,000,000 copies, about1,000,000 copies to about 50,000,000 copies, about 2,000,000 copies toabout 5,000,000 copies, about 2,000,000 copies to about 10,000,000copies, about 2,000,000 copies to about 15,000,000 copies, about2,000,000 copies to about 20,000,000 copies, about 2,000,000 copies toabout 50,000,000 copies, about 5,000,000 copies to about 10,000,000copies, about 5,000,000 copies to about 15,000,000 copies, about5,000,000 copies to about 20,000,000 copies, about 5,000,000 copies toabout 50,000,000 copies, about 10,000,000 copies to about 15,000,000copies, about 10,000,000 copies to about 20,000,000 copies, about10,000,000 copies to about 50,000,000 copies, about 15,000,000 copies toabout 20,000,000 copies, about 15,000,000 copies to about 50,000,000copies, or about 20,000,000 copies to about 50,000,000 copies of theencoding. In some embodiments, the scaffold comprises about 1,000,000copies, about 2,000,000 copies, about 5,000,000 copies, about 10,000,000copies, about 15,000,000 copies, about 20,000,000 copies, or about50,000,000 copies of the encoding. In some embodiments, the scaffoldcomprises at least about 1,000,000 copies, about 2,000,000 copies, about5,000,000 copies, about 10,000,000 copies, about 15,000,000 copies, orabout 20,000,000 copies. In some embodiments, the scaffold comprises atmost about 2,000,000 copies, about 5,000,000 copies, about 10,000,000copies, about 15,000,000 copies, about 20,000,000 copies, or about50,000,000 copies of the encoding.

In some embodiments, the encoding is nucleic acid comprising a barcodesequence. In some embodiments, the encoding comprises a DNA barcode. Insome embodiments, there is at least 1 DNA barcode per bead, at least 10copies of the DNA barcode per bead, at least 100 copies, at least 1,000copies, at least 100,000 copies, at least 1 million copies, or at least10 million copies of the DNA barcode per bead. In some embodiments, thescaffold comprises at least 10 million copies of the DNA barcode perbead.

In some embodiments, DNA barcodes are used to identify a scaffold. Insome instances, the scaffold is a bead. In some instances, only 1 DNAbarcode out of 10 million DNA barcodes is required to identify the bead.In some instances, only 5 DNA barcodes, 10 DNA barcodes, 20 DNAbarcodes, 50 DNA barcodes, 100 DNA barcodes, 1000 DNA barcodes, 10,000DNA barcodes, 100,000 DNA barcodes, or 1 million DNA barcodes out of 10million barcodes is required to identify the bead.

Sample

Samples of any type can be utilized with the methods and systemsprovided herein. In some embodiments, the sample is a biological sample.In some embodiments, the sample comprises one or more cells, one or moreproteins, one or more enzymes, one or more nucleic acids, one or morecellular lysates, or one or more tissue extracts.

In some embodiments, the sample is a cell. In some embodiments, the cellis a eukaryotic cell. In some embodiments, the cell is a prokaryoticcell. In some embodiments, the cell is a mammalian cell. In someembodiments, the cell is a bacterial cell. In some embodiments, the cellis a human cell. In some embodiments, the cell is a cancer cell. In someembodiments, the cell is SH-SYSY, Human neuroblastoma; Hep G2, HumanCaucasian hepatocyte carcinoma; 293 (also known as HEK 293), HumanEmbryo Kidney; RAW 264.7, Mouse monocyte macrophage; HeLa, Human cervixepitheloid carcinoma; MRC-5 (PD 19), Human fetal lung; A2780, Humanovarian carcinoma; CACO-2, Human Caucasian colon adenocarcinoma; THP 1,Human monocytic leukemia; A549, Human Caucasian lung carcinoma; MRC-5(PD 30), Human fetal lung; MCF7, Human Caucasian breast adenocarcinoma;SNL 76/7, Mouse SIM strain embryonic fibroblast; C2C12, Mouse C3H musclemyoblast; Jurkat E6.1, Human leukemic T cell lymphoblast; U937, HumanCaucasian histiocytic lymphoma; L929, Mouse C3H/An connective tissue;3T3 L1, Mouse Embryo; HL60, Human Caucasian promyelocytic leukaemia;PC-12, Rat adrenal phaeochromocytoma; HT29, Human Caucasian colonadenocarcinoma; OE33, Human Caucasian oesophageal carcinoma; OE19, HumanCaucasian oesophageal carcinoma; NIH 3T3, Mouse Swiss NIH embryo;MDA-MB-231, Human Caucasian breast adenocarcinoma; K562, Human Caucasianchronic myelogenous leukemia; U-87 MG, Human glioblastoma astrocytoma;MRC-5 (PD 25), Human fetal lung; A2780cis, Human ovarian carcinoma; B9,Mouse B cell hybridoma; CHO-K1, Hamster Chinese ovary; MDCK, CanineCocker Spaniel kidney; 1321N1, Human brain astrocytoma; A431, Humansquamous carcinoma; ATDCS, Mouse 129 teratocarcinoma AT805 derived; RCC4PLUS VECTOR ALONE, Renal cell carcinoma cell line RCC4 stablytransfected with an empty expression vector, pcDNA3, conferring neomycinresistance.; HUVEC (5200-05n), Human Pre-screened Umbilical VeinEndothelial Cells (HUVEC); neonatal; Vero, Monkey African Green kidney;RCC4 PLUS VHL, Renal cell carcinoma cell line RCC4 stably transfectedwith pcDNA3-VHL; Fao, Rat hepatoma; J774A.1, Mouse BALB/c monocytemacrophage; MC3T3-E1, Mouse C57BL/6 calvaria; J774.2, Mouse BALB/cmonocyte macrophage; PNT1A, Human post pubertal prostate normal,immortalised with SV40; U-2 OS, Human Osteosarcoma; HCT 116, Human coloncarcinoma; MA104, Monkey African Green kidney; BEAS-2B, Human bronchialepithelium, normal; NB2-11, Rat lymphoma; BHK 21 (clone 13), HamsterSyrian kidney; NS0, Mouse myeloma; Neuro 2a, Mouse Albino neuroblastoma;SP2/0-Ag14, Mouse x Mouse myeloma, non-producing; T47D, Human breasttumor; 1301, Human T-cell leukemia; MDCK-II, Canine Cocker SpanielKidney; PNT2, Human prostate normal, immortalized with SV40; PC-3, HumanCaucasian prostate adenocarcinoma; TF1, Human erythroleukaemia; COS-7,Monkey African green kidney, SV40 transformed; MDCK, Canine CockerSpaniel kidney; HUVEC (200-05n), Human Umbilical Vein Endothelial Cells(HUVEC); neonatal; NCI-H322, Human Caucasian bronchioalveolar carcinoma;SK.N. SH, Human Caucasian neuroblastoma; LNCaP.FGC, Human Caucasianprostate carcinoma; OE21, Human Caucasian oesophageal squamous cellcarcinoma; PSN1, Human pancreatic adenocarcinoma; ISHIKAWA, Human Asianendometrial adenocarcinoma; MFE-280, Human Caucasian endometrialadenocarcinoma; MG-63, Human osteosarcoma; RK 13, Rabbit kidney, BVDVnegative; EoL-1 cell, Human eosinophilic leukemia; VCaP, Human ProstateCancer Metastasis; tsA201, Human embryonal kidney, SV40 transformed;CHO, Hamster Chinese ovary; HT 1080, Human fibrosarcoma; PANC-1, HumanCaucasian pancreas; Saos-2, Human primary osteogenic sarcoma; FibroblastGrowth Medium (116K-500), Fibroblast Growth Medium Kit; ND7/23, Mouseneuroblastoma x Rat neuron hybrid; SK-0V-3, Human Caucasian ovaryadenocarcinoma; COV434, Human ovarian granulosa tumor; Hep 3B, Humanhepatocyte carcinoma; Vero (WHO), Monkey African Green kidney; Nthy-ori3-1, Human thyroid follicular epithelial; U373 MG (Uppsala), Humanglioblastoma astrocytoma; A375, Human malignant melanoma; AGS, HumanCaucasian gastric adenocarcinoma; CAKI 2, Human Caucasian kidneycarcinoma; COLO 205, Human Caucasian colon adenocarcinoma; COR-L23,Human Caucasian lung large cell carcinoma; IMR 32, Human Caucasianneuroblastoma; QT 35, Quail Japanese fibrosarcoma; WI 38, HumanCaucasian fetal lung; HMVII, Human vaginal malignant melanoma; HT55,Human colon carcinoma; TK6, Human lymphoblast, thymidine kinaseheterozygote; SP2/0-AG14 (AC-FREE), Mouse x mouse hybridomanon-secreting, serum-free, animal component (AC) free; AR42J, or Ratexocrine pancreatic tumor, or any combination thereof.

In some embodiments, the sample is a protein. In some embodiments, thesample is a recombinant protein. In some embodiments, the sample is amutant protein. In some embodiments, the sample is an enzyme. In someembodiments, the sample is a mutant enzyme. In some embodiments, theenzyme is a protease, a hydrolase, a kinase, a recombinase, a reductase,a dehydrogenase, an isomerase, a synthetase, an oxidoreductase, atransferase, a lyase, a ligase, or any mutant thereof.

In some embodiments, the sample is a single cell. In some embodiments,sample is 2 or more cells. In some embodiments, the sample is at least2, at least 3, at least 4, at least 5, at least 10, at least 100, atleast 1000, or at least 10000 cells.

In some embodiments, the cells comprise transfected nucleic acids. Insome embodiments, the cells comprise stably integrated nucleic acids.

Ion Channel Screen

In some embodiments, the cells comprise ion channels. In someembodiments, the ion channels are endogenous to the cells. In someembodiments, the ion channels are non-endogenous to the cells. In someembodiments, the ion channels are mutant ion channels. In someembodiments, the ion channels comprise a mutation. In some embodiments,the mutation sensitizes the ion channel to optical stimulation. In someembodiments, the optical stimulation is stimulation with electromagneticradiation. In some embodiments, the optical stimulation is stimulationwith visible light.

In some embodiments, the methods comprise stimulating ion channels.Stimulating ion channels may comprise activating or deactivating an ionchannel. In some embodiments, the ion channels are stimulated byelectrostimulation, optical stimulation, or chemical stimulation. Insome embodiments, the stimulation is electrostimulation. In someembodiments, electrostimulation comprises delivering an electric fieldto an ion channel. In some embodiments, the electrostimulation isperformed by an electrode. In some embodiments, the electrostimulationis performed by an electrode on a microfluidic device. In someembodiments, the electrode is within a flow path of a microfluidicdevice. In some embodiments, the electrode is within a flow path of anencapsulation. In some embodiments, the electrode is outside of a flowpath of a microfluidic device. In some embodiments, the electrode isoutside a flow path of an encapsulation.

In some embodiments, provided herein, is a method for screening ionchannel modulators. In some embodiments, the ion channel modulator is aninhibitor. In some embodiments, the ion channel modulator is an agonist.In some embodiments, the method comprises providing an encapsulation. Insome embodiments, the encapsulation comprises a cell expressing an ionchannel protein. In some embodiments, the encapsulation comprises a setof voltage sensor probes. In some embodiments, the encapsulationcomprises an encoded effector and its corresponding encoding. In someembodiments, the encapsulation comprises a cell expressing an ionchannel protein, a set of voltage sensor probes, and an encoded effectorand its corresponding encoding. In some embodiments, the methodcomprises stimulating an ion channel of the cell. In some embodiments,the method comprises detecting a signal from at least one member of theset of voltage sensor probes. In some embodiments, the method comprisessorting the encapsulation. In some embodiments, the method comprisessorting the encapsulation based on the presence, absence, level, orchange of the signal. In some embodiments, the method comprisesmeasuring a property of the encoding to ascertain the identity of theeffector. In some embodiments, the encoding is a nucleic acid and theproperty measured to ascertain the identity of the effector is thenucleic acid sequence of the encoding.

The ion channel protein may be any such protein. In some embodiments,the ion channel protein comprises a sodium, calcium, chloride, proton,or potassium ion channel protein. In some embodiments, the ion channelprotein comprises a sodium ion channel protein. In some embodiments, theion channel protein comprises a potassium ion channel protein. In someembodiments, the ion channel protein comprises a calcium ion channelprotein. In some embodiments, the ion channel protein comprises achloride ion channel protein. In some embodiments, the ion channelprotein comprises a proton ion channel protein.

In some embodiments, the ion channel protein comprises a voltage gatedion channel protein. Any voltage gated ion channel protein may be used.In some embodiments, the voltage gated ion channel protein comprises asodium, calcium, chloride, proton, or potassium voltage gated ionchannel protein. In some embodiments, the voltage gated ion channelprotein comprises a voltage gated calcium ion channel protein. In someembodiments, the voltage gated ion channel protein comprises a voltagegated sodium ion channel protein. In some embodiments, the voltage gatedion channel protein comprises a voltage gated potassium ion channelprotein. In some embodiments, the voltage gated ion channel proteincomprises a voltage gated chloride ion channel protein. In someembodiments, the voltage gated ion channel protein comprises a voltagegated proton ion channel protein.

In some embodiments, the ion channel protein is endogenous to the cell.In some embodiments, the ion channel protein is an exogenous ion channelprotein. In some embodiments, the ion channel protein is incorporatedinto the cell through a vector. In some embodiments, the ion channelprotein stably expressed in the cell through the addition of a vector.In some embodiments, a gene encoding the ion channel protein istransiently transfected into the cell. In some embodiments, a geneencoding the ion channel is stably incorporated into the cell. In someembodiments, the ion channel protein is overexpressed.

In some embodiments, the voltage gated ion channel protein comprises avoltage-gated calcium channel protein (VGCC). Any VGCC or any mutant,fragment, or conjugate thereof may be used. In some embodiments, theVGCC comprises an L-type calcium channel (e.g. Ca_(v)1.1, Ca_(v)1.2,Ca_(v)1.3, or Ca_(v)1.4), a P-type calcium channel (e.g. Ca_(v)2.1), anN-type calcium channel (e.g. Ca_(v)2.2), an R-type calcium channel (e.g.Ca_(v)2.3), or a T-type calcium channel (e.g. Ca_(v)3.1, Ca_(v)3.2, orCa_(v)3.3), or any mutant, fragment, or conjugate thereof. In someembodiments, the VGCC comprises an L-type calcium channel. In someembodiments, the VGCC comprises a P-type calcium channel. In someembodiments, the VGCC comprises an N-type calcium channel. In someembodiments, the VGCC comprises an R-type calcium channel. In someembodiments, the VGCC comprises a T-type calcium channel.

In some embodiments, the ion channel protein comprises a voltage gatedsodium channel protein (Nay) or any mutant, fragment, or conjugatethereof. Any voltage gated sodium channel protein may be used. In someembodiments, the voltage gated sodium channel protein comprisesNa_(v)1.1, Na_(v)1.2, Na_(v)1.3, Na_(v)1.4, Na_(v)1.5, Na_(v)1.6,Na_(v)1.7, Navl. 8, Na_(v)1.9, Na_(v)2.1, Na_(v)2.2, Na_(v)2.3, orNa_(v)3.1, or any mutant, fragment, or conjugate thereof.

In some embodiments, the ion channel protein comprises a voltage gatedpotassium channel protein (VGKC) or any mutant, fragment, or conjugatethereof. Any VGKC protein may be used. The VGKC protein may have anyalpha subunit. In some embodiments, the VGKC comprises a delayedrectifier potassium channel (e.g. K_(v)1.1, K_(v)1.2, K_(v)1.3,K_(v)1.5, K_(v)1.6, K_(v)1.7, K_(v)1.8, K_(v)2.1, K_(v)2.1, K_(v)3.1,K_(v)3.2, K_(v)7.1, K_(v)7.2, K_(v)7.3, K_(v)7.4, K_(v)7.5, orK_(v)10.1). In some embodiments, the VGKC comprises an A-type potassiumchannel (E.g. K_(v)1.4, K_(v)3.3, K_(v)3.4, K_(v)4.1, K_(v)4.1,K_(v)4.2, or K_(v)4.3). In some embodiments, the VGKC comprises anoutward-rectifying potassium channel (e.g. K_(v)10.2). In someembodiments, the VGKC comprises an inwardly-rectifying potassium channel(e.g. an ether-a-go-go potassium channel, such as K_(v)11.1, K_(v)11.2,or K_(v)11.3). In some embodiments, the VGKC comprises a slowlyactivating potassium channel (e.g. K_(v)12.1, K_(v)12.2, or K_(v)12.3).In some embodiments, the VGKC comprises a modifier/silencer potassiumchannel (e.g. K_(v)5.1, K_(v)6.1, K_(v)6.2, K_(v)6.3, K_(v)6.4,K_(v)8.1, K_(v)8.2, K_(v)9.1, K_(v)9.2, or K_(v)9.3). Any mutant,fragment, or conjugate of any of the preceding potassium channels may beused.

In some embodiments, the ion channel protein comprises a voltage gatedchloride channel protein. Any voltage gated chloride channel protein maybe used. In some embodiments, the voltage gated chloride channel proteinis from the CLCN family (e.g. CLCN1, CLCN2, CLCN3, CLCN4, CLCN5, CLCN6,CLCN7, CLCNKA, CLCNKB). In some embodiments, the voltage gated chloridechannel protein is from the epithelial chloride channel family (e.g.CLCA1, CLCA2, CLCA3, or CLCA4). In some embodiments, the voltage gatedchloride channel protein is from the chloride intracellular channel(CLIC) family (e.g. CLIC1, CLIC2, CLIC3, CLIC4, CLIC5, or CLIC6).

In some embodiments, the ion channel protein comprises a voltage gatedproton channel. Any voltage gated proton channel protein may be used. Insome embodiments, the voltage gated proton channel comprisevoltage-gated hydrogen channel 1 protein.

In some embodiments, the ion channel protein comprises achannelrhodopsin or any mutant, fragment, or conjugate thereof. In someembodiments, wherein the channelrhodopsin is ChrimsonR or any mutant,fragment, or conjugate thereof. In some embodiments, thechannelrhodopsin is a ChrimsonR mutant comprising a K176R mutation,S267M mutation, Y268F mutation, Y261F mutation, or any combinationthereof.

The set of voltage sensor probes may comprise any suitable probe. Insome embodiments, the set of voltage sensor probes comprise a FRET pair.In some embodiments, the set of voltage sensor probes comprises avoltage-sensitive oxonol, a fluorescent coumarin, or both. In someembodiments, the set of voltage sensor probes comprises avoltage-sensitive oxonol. In some embodiments, the set of voltage sensorprobes comprises a fluorescent coumarin. In some embodiments, the set ofvoltage sensor probes comprises a DiSBAC compound, a coumarinphospholipid, or any combination or derivative thereof. In someembodiments, the set of voltage sensor probes comprises a DiSBACcompound. In some embodiments, the set of voltage sensor probescomprises a coumarin phospholipid. the set of voltage sensors comprisesa DiSBAC₂, DiSBAC₄, DiSBAC₆, CC1-DMPE, CC2-DMPE, or any combination orderivative thereof. In some embodiments, the set of voltage sensorscomprises a DiSBAC₂(3), DiSBAC₂(5), DiSBAC₄(3), DiSBAC₄(5), DiSBAC₆(3),DiSBAC₆(5), CC1-DMPE, CC2-DMPE, or any combination or derivativethereof. In some embodiments, the set of voltage sensors comprisesDiSBAC₆ and CC2-DMPE.

The encapsulation may further comprise a voltage assay backgroundsuppression compound. In some embodiments, the voltage assay backgroundsuppression compound comprises VABSC-1.

In some embodiments, the stimulation is optical stimulation. In someembodiments, the optical stimulation is electromagnetic radiation. Insome embodiments, the optical stimulation is visible light. In someembodiments, the optical stimulation is UV, VIS, or near-infraredradiation. In some embodiments, the optical stimulation is UV radiation.In some embodiments, the optical stimulation is visible light. In someembodiments, the optical stimulation is near-infrared radiation.

In some embodiments, the wavelength of light for optical stimulation isabout 660 nm. In some embodiments, the wavelength of light for opticalstimulation is about 100 nm to about 1,000 nm. In some embodiments, thewavelength of light for optical stimulation is about 100 nm to about 200nm, about 100 nm to about 400 nm, about 100 nm to about 450 nm, about100 nm to about 500 nm, about 100 nm to about 550 nm, about 100 nm toabout 600 nm, about 100 nm to about 650 nm, about 100 nm to about 700nm, about 100 nm to about 750 nm, about 100 nm to about 800 nm, about100 nm to about 1,000 nm, about 200 nm to about 400 nm, about 200 nm toabout 450 nm, about 200 nm to about 500 nm, about 200 nm to about 550nm, about 200 nm to about 600 nm, about 200 nm to about 650 nm, about200 nm to about 700 nm, about 200 nm to about 750 nm, about 200 nm toabout 800 nm, about 200 nm to about 1,000 nm, about 400 nm to about 450nm, about 400 nm to about 500 nm, about 400 nm to about 550 nm, about400 nm to about 600 nm, about 400 nm to about 650 nm, about 400 nm toabout 700 nm, about 400 nm to about 750 nm, about 400 nm to about 800nm, about 400 nm to about 1,000 nm, about 450 nm to about 500 nm, about450 nm to about 550 nm, about 450 nm to about 600 nm, about 450 nm toabout 650 nm, about 450 nm to about 700 nm, about 450 nm to about 750nm, about 450 nm to about 800 nm, about 450 nm to about 1,000 nm, about500 nm to about 550 nm, about 500 nm to about 600 nm, about 500 nm toabout 650 nm, about 500 nm to about 700 nm, about 500 nm to about 750nm, about 500 nm to about 800 nm, about 500 nm to about 1,000 nm, about550 nm to about 600 nm, about 550 nm to about 650 nm, about 550 nm toabout 700 nm, about 550 nm to about 750 nm, about 550 nm to about 800nm, about 550 nm to about 1,000 nm, about 600 nm to about 650 nm, about600 nm to about 700 nm, about 600 nm to about 750 nm, about 600 nm toabout 800 nm, about 600 nm to about 1,000 nm, about 650 nm to about 700nm, about 650 nm to about 750 nm, about 650 nm to about 800 nm, about650 nm to about 1,000 nm, about 700 nm to about 750 nm, about 700 nm toabout 800 nm, about 700 nm to about 1,000 nm, about 750 nm to about 800nm, about 750 nm to about 1,000 nm, or about 800 nm to about 1,000 nm.In some embodiments, the wavelength of light for optical stimulation isabout 100 nm, about 200 nm, about 400 nm, about 450 nm, about 500 nm,about 550 nm, about 600 nm, about 650 nm, about 700 nm, about 750 nm,about 800 nm, or about 1,000 nm. In some embodiments, the wavelength oflight for optical stimulation is at least about 100 nm, about 200 nm,about 400 nm, about 450 nm, about 500 nm, about 550 nm, about 600 nm,about 650 nm, about 700 nm, about 750 nm, or about 800 nm. In someembodiments, the wavelength of light for optical stimulation is at mostabout 200 nm, about 400 nm, about 450 nm, about 500 nm, about 550 nm,about 600 nm, about 650 nm, about 700 nm, about 750 nm, about 800 nm, orabout 1,000 nm.

In some embodiments, the intensity of light for optical stimulation isabout 500 mJ/s/cm². In some embodiments, intensity of light for opticalstimulation is about 50 to about 1,000 mJ/S/cm². In some embodiments,intensity of light for optical stimulation is about 50 to about 100,about 50 to about 250, about 50 to about 500, about 50 to about 750,about 50 to about 1,000, about 100 to about 250, about 100 to about 500,about 100 to about 750, about 100 to about 1,000, about 250 to about500, about 250 to about 750, about 250 to about 1,000, about 500 toabout 750, about 500 to about 1,000, or about 750 to about 1,000mJ/S/cm². In some embodiments, intensity of light for opticalstimulation is about 50, about 100, about 250, about 500, about 750, orabout 1,000 mJ/S/cm². In some embodiments, intensity of light foroptical stimulation is at least about 50, about 100, about 250, about500, or about 750. In some embodiments, intensity of light for opticalstimulation is at most about 100, about 250, about 500, about 750, orabout 1,000 mJ/S/cm².

In some embodiments, the frequency of optical stimulation is about 10Hz. In some embodiments, the frequency of optical stimulation is about 1Hz to about 100 Hz. In some embodiments, the frequency of opticalstimulation is about 1 Hz to about 2 Hz, about 1 Hz to about 5 Hz, about1 Hz to about 10 Hz, about 1 Hz to about 20 Hz, about 1 Hz to about 50Hz, about 1 Hz to about 100 Hz, about 2 Hz to about 5 Hz, about 2 Hz toabout 10 Hz, about 2 Hz to about 20 Hz, about 2 Hz to about 50 Hz, about2 Hz to about 100 Hz, about 5 Hz to about 10 Hz, about 5 Hz to about 20Hz, about 5 Hz to about 50 Hz, about 5 Hz to about 100 Hz, about 10 Hzto about 20 Hz, about 10 Hz to about 50 Hz, about 10 Hz to about 100 Hz,about 20 Hz to about 50 Hz, about 20 Hz to about 100 Hz, or about 50 Hzto about 100 Hz. In some embodiments, the frequency of opticalstimulation is about 1 Hz, about 2 Hz, about 5 Hz, about 10 Hz, about 20Hz, about 50 Hz, or about 100 Hz. In some embodiments, the frequency ofoptical stimulation is at least about 1 Hz, about 2 Hz, about 5 Hz,about 10 Hz, about 20 Hz, or about 50 Hz. In some embodiments, thefrequency of optical stimulation is at most about 2 Hz, about 5 Hz,about 10 Hz, about 20 Hz, about 50 Hz, about 100 Hz, about 150 Hz, orabout 200 Hz.

In some embodiments, stimulation is chemical stimulation. In someembodiments, the chemical stimulation comprises contacting the ionchannel with a toxin. In some embodiments, the toxin is an ion channeltoxin. In some embodiments, the toxin is added to an encapsulation bypico-injection. In some embodiments, the toxin is added to anencapsulation by conditional pico-injection. In some embodiments,chemical stimulation comprises contacting the ion channel with an ionchannel toxin. In some embodiments, the ion channel toxin comprisesveratridine, OD-1, or another ion channel toxin, or any combinationthereof. In some embodiments, the ion channel toxin comprisesveratridine. In some embodiments, the ion channel toxin comprises OD-1.

In some embodiments, the ion channel toxin as added to the encapsulationby pico-injection, droplet fusion, or through a pre-arrangedarchitecture of a microfluidic device which contains the encapsulation.In some embodiments, the ion channel toxin as added to the encapsulationby pico-injection. In some embodiments, the ion channel toxin as addedto the encapsulation by droplet fusion. In some embodiments, the ionchannel toxin as added to the encapsulation through a pre-arrangedarchitecture of a microfluidic device which contains the encapsulation.

The ion channel may be stimulated by electrical stimulation. In someembodiments, stimulating the ion channel is performed by at least oneelectrode. In some embodiments, the at least one electrode is in theflow path of the encapsulation. In some embodiments, the at least oneelectrode is outside the flow path of the encapsulation. In someembodiments, electrostimulation is performed by non-contact electrodesto generate electric fields, dielectrophoretic forces, or embeddedmetal-contact electrodes. In some embodiments, electrostimulation isperformed by non-contact electrodes to generate electric fields. In someembodiments, electrostimulation is performed dielectrophoretic forces.In some embodiments, electrostimulation is performed by embeddedmetal-contact electrodes.

In some embodiments, electrostimulation is dictated by geometry of amicrofluidic device containing the encapsulation. In some embodiments,the frequency of electrostimulation is about 10 Hz. In some embodiments,the frequency of electrostimulation is about 1 Hz to about 100 Hz. Insome embodiments, the frequency of electrostimulation is about 1 Hz toabout 2 Hz, about 1 Hz to about 5 Hz, about 1 Hz to about 10 Hz, about 1Hz to about 20 Hz, about 1 Hz to about 50 Hz, about 1 Hz to about 100Hz, about 2 Hz to about 5 Hz, about 2 Hz to about 10 Hz, about 2 Hz toabout 20 Hz, about 2 Hz to about 50 Hz, about 2 Hz to about 100 Hz,about 5 Hz to about 10 Hz, about 5 Hz to about 20 Hz, about 5 Hz toabout 50 Hz, about 5 Hz to about 100 Hz, about 10 Hz to about 20 Hz,about 10 Hz to about 50 Hz, about 10 Hz to about 100 Hz, about 20 Hz toabout 50 Hz, about 20 Hz to about 100 Hz, or about 50 Hz to about 100Hz. In some embodiments, the frequency of electrostimulation is about 1Hz, about 2 Hz, about 5 Hz, about 10 Hz, about 20 Hz, about 50 Hz, orabout 100 Hz. In some embodiments, the frequency of electrostimulationis at least about 1 Hz, about 2 Hz, about 5 Hz, about 10 Hz, about 20Hz, or about 50 Hz. In some embodiments, the frequency ofelectrostimulation is at most about 2 Hz, about 5 Hz, about 10 Hz, about20 Hz, about 50 Hz, about 100 Hz, about 150 Hz, or about 200 Hz.

The stimulation of the ion channels can be performed numerous times, oronly a single time. In some embodiments, the ion channel of the cell isstimulated about 1 time to about 20 times. In some embodiments, the ionchannel of the cell is stimulated about 1 time to about 2 times, about 1time to about 3 times, about 1 time to about 5 times, about 1 time toabout 7 times, about 1 time to about 10 times, about 1 time to about 20times, about 2 times to about 3 times, about 2 times to about 5 times,about 2 times to about 7 times, about 2 times to about 10 times, about 2times to about 20 times, about 3 times to about 5 times, about 3 timesto about 7 times, about 3 times to about 10 times, about 3 times toabout 20 times, about 5 times to about 7 times, about 5 times to about10 times, about 5 times to about 20 times, about 7 times to about 10times, about 7 times to about 20 times, or about 10 times to about 20times. In some embodiments, the ion channel of the cell is stimulatedabout 1 time, about 2 times, about 3 times, about 5 times, about 7times, about 10 times, or about 20 times. In some embodiments, the ionchannel of the cell is stimulated at least about 1 time, about 2 times,about 3 times, about 5 times, about 7 times, or about 10 times. In someembodiments, the ion channel of the cell is stimulated at most about 2times, about 3 times, about 5 times, about 7 times, about 10 times, orabout 20 times. In some embodiments, the ion channel is stimulated asingle time. In embodiments where stimulation occurs by the addition ofan ion channel toxin or other ion channel inhibitor, the ion channeltoxin need only be added at a single step.

In some embodiments, provided herein, are methods for stimulating an ionchannel. In some embodiments, the methods comprise providing a cell inan encapsulation. In some embodiments, the methods comprise stimulatingan ion channel of the cell by electrostimulation, optical stimulation,or chemical stimulation. In some embodiments, the methods comprisedetecting a signal from the cell by capturing images of the cell in theencapsulation.

In some embodiments, the method comprises detecting a signal from atleast one member of the set of voltage sensor probes. In someembodiments, the signal is electromagnetic radiation. In someembodiments, the electromagnetic radiation is luminescence orfluorescence. In some embodiments, the electromagnetic radiation isfluorescence. In some embodiments, the electromagnetic radiation isemitted due to a FRET interaction. In some embodiments, the signal is anincrease, decrease, or change in electromagnetic radiation as comparedto an identical encapsulation without the encoded effector. In someembodiments, the signal is an increase, decrease, or change inelectromagnetic radiation as compared to the encapsulation before thestimulation of the ion channel.

In some embodiments, the method comprises the step of sorting theencapsulation based on the presence, absence, level, or change of thesignal. In some embodiments, the method further comprises measuring aproperty of the encoding to ascertain the identity of the effector.

In some embodiments, the sample is a protein. In some embodiments, thesample is a recombinant protein. In some embodiments, the sample is amutant protein. In some embodiments, the sample is an enzyme. In someembodiments, the sample is a mutant enzyme. In some embodiments, theenzyme is a protease, a hydrolase, a kinase, a recombinase, a reductase,a dehydrogenase, an isomerase, a synthetase, an oxidoreductase, atransferase, a lyase, a ligase, or any mutant thereof.

The sample may further comprise a nucleic acid which codes for theexpression of a target protein and the target protein itself. Thesesample nucleic acids may be barcoded. The presence of a barcode on thenucleic acids may allow for the transfer of the barcode to nucleic acidencodings of effectors that are co-encapsulated with the target proteinand the nucleic acid which codes for the expression of the targetprotein. This in turn allows for a determination of which combinationsof effectors were encapsulated together and produced a synergisticeffect against the target protein. Such methods can be used to conductfragment-based screens to identify lead molecules of interest in furtherdrug discovery.

Fragment Based Screen and Enzyme Evolution Method

In some embodiments, the sample is a target protein and a nucleic acidcoding the expression of a target protein. In some embodiments, thenucleic acid coding the expression of the target protein furthercomprises a barcode region. In some embodiments, the nucleic acid codingthe expression of a target protein is bound to a scaffold. In someembodiments, the barcode from the nucleic acid that codes for the targetprotein can be transferred to nucleic acid encodings of effectors. Insome embodiments, the sample target protein and nucleic acid coding theexpression of the target protein are co-encapsulated with an in vitrotranscription/translation system. In some embodiments, the in vitrotranscription/translation system is used to amplify the target protein.

In some embodiments, two or more nucleic acid encoded effectors withtheir corresponding nucleic acid encodings are introduced into theencapsulation comprising the target protein and nucleic acid encodingthe expression of the target protein. In some embodiments, the barcodeis transferred to the nucleic acids encoding the effectors. In someembodiments, the encapsulation is incubated for a period of time toallow the two or more effectors to interact with the target protein. Insome embodiments, a signal is produced by the interaction of the two ormore effectors and the target protein. In some embodiments, theencapsulation is sorted based on the measurement of the signal. In someembodiments, the nucleic acid encodings which now comprise the barcodefrom nucleic acid coding for the target protein are sequenced. In someembodiments, the sequencing allows for identifying combinations ofeffectors that conferred efficacy against the target protein.

In some embodiments, the target protein coded by the nucleic acid is asignaling protein, an enzyme, a binding protein, an antibody or antibodyfragment, a structure protein, a storage protein, or a transportprotein. In some embodiments, the target protein is an enzyme. In someembodiments, the target protein is trypsin, macrophage metalloelastase12 (MMP-12), extracellular signal-related kinase 1 (ERK1), orextracellular signal-regulated kinase 2 (EKR2).

In embodiments wherein the sample is a target protein and a nucleic acidcoding the expression of the target protein, the nucleic acid maycomprise a sequence complementary to the nucleic acid encoding aneffector. This complementarity can be utilized for amplification of thebarcode onto the nucleic acid encoding the effector. In embodimentswherein the sample is a target protein and a nucleic acid coding theexpression of the target protein, the nucleic acid may contain apromoter sequence. In some embodiments, the promoter sequence allows foramplification of the nucleic acid sequence and/or the nucleic acidsequence encoding the effector after the barcode has been transferred.

In vitro transcription/translation systems are systems which can expressproteins from nucleic acids which code for the protein without requiringany living tissue or cells. In some embodiments, the in vitrotranscription/translation system is used to express the target proteinwithin an encapsulation. In some embodiments, the in vitrotranscription/translation system is used to express the target proteinwithin an encapsulation to a target concentration. In some embodiments,the in vitro transcription/translation system is used to amplify thetarget protein within an encapsulation. In some embodiments, the invitro transcription/translation system is used to amplify the targetprotein within an encapsulation to a desired concentration.

Encapsulation

An encapsulation can refer to the formation of a compartment within alarger system. In preferred embodiments, the encapsulation is a dropletwithin a microfluidic channel. In some embodiments, the encapsulation isa droplet, an emulsion, a macrowell, a microwell, bubble, or amicrofluidic confinement. Once an encapsulation is formed, any componentinside the encapsulation can remain in the encapsulation until theencapsulation is destroyed or broken down. In some embodiments, theencapsulations used herein remain stable for at least 4 hours, at least12 hours, at least 1 day, at least 2 days, at least 3 days, or at least1 week. In some embodiments, the encapsulations are stable for theduration of the screen to be performed so that no intermingling ofreagents between encapsulations occurs.

In some embodiments, the encapsulation is a droplet. In someembodiments, the droplet is at most 1 picoliter, at most 10 picoliters,at most 100 picoliters, at most 1 nanoliter, at most 10 nanoliters, atmost 100 nanoliters, or at most 1 microliter in volume. In someembodiments, the droplet is at least 1 picoliter, at least 10picoliters, at least 100 picoliters, at least 1 nanoliter, at least 10nanoliters, at least 100 nanoliters, or at least 1 microliter in volume.In some embodiments, the droplet is between about 200 picoliters andabout 10 nanoliters.

In some embodiments, the droplet is an aqueous droplet in a larger bodyof oil. In some embodiments, the droplets are placed in an oil emulsion.In some embodiments, the oil comprises a silicone oil, a fluorosiliconeoil, a hydrocarbon oil, a mineral oil, a paraffin oil, a halogenatedoil, a fluorocarbon oil, or any combination thereof. In someembodiments, the oil comprises a silicone oil. In some embodiments, theoil comprises a fluorosilicone oil. In some embodiments, the oilcomprises a hydrocarbon oil. In some embodiments, the oil comprises amineral oil. In some embodiments, the oil comprises a paraffin oil. Insome embodiments, the oil comprises a halogenated oil. In someembodiments, the oil comprises a fluorocarbon oil.

In embodiments wherein there are a plurality of encapsulations, eachindividual encapsulation may be any size. In some embodiments, eachencapsulation is approximately the same size. In some embodiments, eachencapsulation is within 5%, 10%, 15%, 20%, or 25% of the average sizeencapsulation within the plurality. In some embodiments, at least 80%,85%, 90%, or 95% of the encapsulations are within about 5%, 10%, 15%,20%, or 25% of the average size encapsulation within the plurality.

The encapsulations may be formed by any method. In some embodiments, anencapsulation is formed by flowing an aqueous stream into an immisciblecarrier fluid. In some embodiment, the aqueous stream flows into animmiscible carrier fluid at a junction of microfluidic channels. In someembodiments, the junction is a T-junction. In some embodiments, thejunction is a meeting of two perpendicular microfluidic channels. Thejunction may be a meeting of any number of microfluidic channels. Thejunction may be at any angle. The aqueous stream may be formed by anupstream junction of two or more aqueous streams. In some embodiments,sample solutions and effector solutions are joined upstream of theaqueous stream junction with the immiscible carrier fluid.

The size of the droplets may be controlled by modulating a variety ofparameters. These parameters include the geometry of the junction of twomicrofluidic channels, the flow rate of the two streams, the type of oilused, the presence of surfactants, the pressure applied to the flowstreams, or any combination thereof.

In some embodiments, a single encoded effector is present in anencapsulation. In some embodiments, a single scaffold comprising anencoded effector and its encoding are present in an encapsulation. Insome embodiments, a plurality of scaffolds, each scaffold comprising adifferent encoded effector and its respective encoding, are present inan encapsulation.

In some embodiments, encapsulations comprise biological samples. In someembodiments, encapsulations comprise single cells. In some embodiments,encapsulations comprise one or more cells. In some embodiments, theencapsulations comprise nucleic acids. In some embodiments, theencapsulations comprise proteins. In some embodiments, theencapsulations comprise.

Sorting

The methods and systems provided herein may comprise sorting steps. Thesorting step can be accomplished in a variety of ways. One way ofsorting the “hit” effectors from the non-hit effectors is to physicallyseparate the hits from non-hits in space. This can be accomplished in avariety of manners. In some embodiments, sorting the encapsulationscomprises providing the encapsulation through a microfluidic channel. Insome embodiments, the microfluidic channel is equipped with a detector.In some embodiments, the “hit” effectors are placed into one collectionvessel if the “hit” criteria is met, and the “non-hit” effectors areplaced into another collection vessel. As described herein, in someembodiments, such “hit” effectors are sorted based on the presence orabsence of a signal resulting from an interaction with the effector (oranother component) and the sample, a reagent, or combinations thereof.In some embodiments, the sorting is based on the level of a signaldetected. In some embodiments, the sorting is based on the presence of asignal detected. In some embodiments, the sorting is based on theabsence of a signal.

In some embodiments, sorting droplets is accomplished by activity-basedscreening. Activity based sorting is accomplished by the ability to sortbased on detecting a response emitted by the droplet as it passes by adetecting region on the microfluidic chip. As an example, certainsmall-molecules inhibit particular enzymes which can be screened by anactivity-based assay that detects for that inhibition. Thus, sorting isbased on the “activity” of the enzyme and thus screening forsmall-molecules that functionally inhibit the enzyme rather than simplybind to the enzyme. It is a more relevant screen and is much moresimilar to conventional HTS screening which screens for activity.

In some embodiments, sorting the encapsulations comprises placing theencapsulations (e.g., droplets) into a first collection tube if thesignal is at or above a predetermine threshold. In some embodiments,sorting the encapsulation comprises placing the droplet into a secondcollection tube if the signal is below a predetermined threshold. Insome embodiments, sorting the encapsulation comprises placing thedroplet into a first collection tube if the signal is at or above apredetermine threshold or placing the droplet into a second collectiontube if the signal is below a predetermined threshold. In someembodiments, sorting the encapsulation comprises placing encapsulationsin two or more collection tubes, or bins. In some embodiments, “hit”effectors or positive “hits’ are stored in two or more collection tubesor bins. In some embodiments, the “hit” effectors, or positive “hits”are sorted based on the signal or activity measured.

FIGS. 26A to 28C depict sorting droplets based on two types of detectionsignals. FIGS. 26A-B depict the use of a bead attached with fluorophoreTR1-TAMRA, which upon release from the bead, provides a detectableintensity level (FIG. 26B). By contrast, FIGS. 27A-B depict the use of abead attached with an inhibitor TR3, which upon release inhibits orminimizes the intensity of fluorescence detected (FIG. 27B). FIG. 27Cdepicts a decrease in Cathepsin D activity with increasing concentrationof the TR3 inhibitor. FIG. 28A provides an exemplary depiction ofdroplets being sorted based on a certain inhibition threshold being met,wherein for those droplets exhibiting a fluorescence intensity levelbelow a certain threshold will be a “positive” hit, and those dropletsexhibiting fluorescence intensity levels above the threshold, will be a“negative” hit. FIG. 28C provides an exemplary threshold level for suchinhibitory activity. In some embodiments, the threshold for sorting willbe based on a minimum fluorescence intensity level being measured (e.g.,as occurring through use of TAMRA fluorophore). FIG. 28B provides anexemplary threshold level for such fluorescence detection activity. FIG.28D provides an exemplary illustration of a device as used in a methodor system described herein.

In some embodiments, sorting the encapsulation comprises using awaveform pulse generator to move the encapsulation to a collection tubeby an electrical field gradient, by sound, by a diaphragm, by modifyinggeometry of microfluidic channel, or by changing the pressure of themicrofluidic channel. In some embodiments, the waveform pulse generatormoves the encapsulation by an electrical field gradient. In someembodiments, the waveform pulse generator moves the encapsulation bysound. In some embodiments, the waveform pulse generator moves theencapsulation by a diaphragm. In some embodiments, the waveform pulsegenerator modifies the geometry of the microfluidic channel. In someembodiments, the waveform pulse generator changes the pressure of themicrofluidic channel.

Various methods for determining which effectors had the desired effectmay be used. In some instances, physical sorting of “hit” effectors isused to determine which effectors had the desired effect. In someinstances, selective addition of a detectable label to encapsulationscomprising a “hit” effector is used. In some instances, a detectablelabel is used to determine which effectors had the desired effect bylinking detectable label with the encoding. For example, the addition ofa nucleic acid barcode to nucleic acid encodings of effectors canaccomplish tagging the “hit” effectors in a way that can be ascertainedby sequencing. If only “hit” effectors encodings are tagged with thenucleic acid barcode, then these samples can be picked out during asubsequent sequencing step, as effectors which lacked the desiredactivity will lack the barcode. The barcode may additionally comprise aunique primer sequence to allow for amplification of only the “hit”effector encodings. In this way, all encapsulations can be pooledtogether, regardless of activity or efficacy, and the resulting hits canstill be ascertained.

Barcode Non-Sorting Method

In some embodiments provided herein, the methods do not comprise aphysical sorting step. In these embodiments, deconvolution of whicheffectors had the desired effect on a sample is accomplished in adifferent manner. In some embodiments, the method further comprises thestep of adding additional reagents to the encapsulation which add abarcode to the encoding. In some embodiments, the method furthercomprises the step of adding additional reagents to the encapsulationwhich add a barcode to a nucleic acid encoding. In some embodiments, theadditional reagents add a barcode to the encoding by annealing thebarcode to the encoding, ligating the barcode to the encoding, oramplifying the barcode onto the encoding. In some embodiments, theadditional reagents comprise a tagging nucleic acid comprising asequence complementary to a sequence on the nucleic acid encoding whichacts as a primer for the nucleic acid encoding and the barcode. In someembodiments, the additional reagents comprise enzymes to add the barcodeto the nucleic acid encoding.

Provided herein, in some embodiments, are methods for screening anencoded effector without a physical sorting step. In some embodiments,the method comprises providing a sample, a nucleic acid encodedeffector, and a nucleic acid encoding in an encapsulation. In someembodiments, a signal is detected in the encapsulation. In someembodiments, the signal results from an interaction between the effectorand the sample. In some embodiments, a first capping mix is added to thedroplet based on the detection, absence, or level of the signal. In someembodiments, the first capping mix adds a first nucleic acid cap to thenucleic acid encoding. In some embodiments, a second capping mix isadded to the encapsulation. In some embodiments, the second capping mixis only added if the first capping mix is not added to theencapsulation. In some embodiments, the first nucleic acid cap and thesecond nucleic acid cap have different sequences. In some embodiments,only the first nucleic acid cap or only the second nucleic acid cap isadded to the nucleic acid encoding.

The first and second nucleic acid caps can have different significanceand indicate different things when added to nucleic acid encodings. Insome embodiments, the first nucleic acid cap indicates that the effectorhad a desired activity. In some embodiments, the desired activityresulted in the signal being above a pre-determined threshold. In someembodiments, the desired activity resulted in the signal being below apre-determined threshold. In some embodiments, the desired activityresulted in the presence of the signal. In some embodiments, the desiredactivity resulted in the absence of the signal.

In some embodiments, the second nucleic acid cap indicates that theeffector lacked a desired activity. In some embodiments, the lack ofdesired activity resulted in the signal being below a pre-determinedthreshold. In some embodiments, the lack of desired activity resulted inthe signal being above a pre-determined threshold. In some embodiments,the lack of desired activity resulted in the absence of the signal. Insome embodiments, the lack of desired activity resulted in the presenceof the signal.

The nucleic acid caps can be added to nucleic acid encodings by avariety of methods. In some embodiments, the nucleic acid cap is addedto the nucleic acid encoding by ligation, hybridization, extension ofthe nucleic acid encoding, or combinations thereof. In some embodiments,the nucleic acid cap is added to the nucleic acid encoding by ligation.In some embodiments, the nucleic acid cap is added to the nucleic acidencoding by hybridization. In some embodiments, the nucleic acid cap isadded to the nucleic acid encoding by extension of the nucleic acidencoding. In some embodiments, the nucleic acid cap is added to thenucleic acid encoding by chemically crosslinking the nucleic acids. Insome embodiments, the nucleic acid cap is added to the nucleic acidencoding by chemical crosslinking with psoralen. In some embodiments, acomplementary sequence the nucleic acid cap is located on the terminalend of the nucleic acid encoding to allow for the addition of thenucleic acid cap. In some embodiments, the nucleic acid caps comprise abarcode sequence.

In some embodiments, the capping mix comprises additional reagents foradding the nucleic acid cap to the encoding. In some embodiments, theadditional reagents comprise an enzyme. In some embodiments, the enzymeis a polymerase, a ligase, a restriction enzyme, or a recombinase. Insome embodiments, the enzyme is a polymerase.

Bead Capture of Nucleic Acids

In addition to measuring activity from detectable signals, additionalinformation can be gathered from a screen by incorporating nucleic acidsfrom the sample onto encodings. In some embodiments, the methodcomprises transferring one or more nucleic acids from the sample to theencoding. The transfer of nucleic acids from the sample to the encodingallows substantial information about the sample, and information aboutthe effect the effector has on the sample to be ascertained,particularly when the sample is a cell. The transfer of the nucleicacids from the sample can allow for quantification of expressed proteinby quantifying the amount of target mRNA, as well as provide globalproteomic and genomic data about the cell. This data can be collectedand compared to cells that did not receive a dose of the indicatedeffector

In one aspect, provided herein, is a method for detecting sample nucleicacids in a nucleic acid encoded effector screen. In some embodiments,the method comprises providing one or more cells, a nucleic acid encodedeffector, and a nucleic acid encoding in an encapsulation. In someembodiments, the encapsulation is incubated for a period of time toallow for the effector and the cell to interact. In some embodiments, asdescribed herein, the interaction between the effector and the cellproduces a signal. In some embodiments, the period of time is sufficientto allow for changes in transcription and/or translation to occur in thecell in response to the effector. In some embodiments, the methodcomprises transferring cellular nucleic acids to the nucleic acidencoding. In some embodiments, the cellular nucleic acids are quantifiedby sequencing the nucleic acid encodings after the cellular nucleicacids have been transferred. In this way, an expression fingerprint ofthe cell can be generated in response to treatment with the effector. Asdescribed herein, in some embodiments, the method further comprisesdetecting a signal produced through interaction between the effector andone or more cells, and sorting the encapsulation based on the detectionof the signal.

In order to release the cellular nucleic acids, the cell may be lysed.In some embodiments, the method further comprises the step of lysing thecell. In some embodiments, lysing the cell comprises adding lysis bufferto the encapsulation. In some embodiments, the lysis buffer is added bypico-injection. In some embodiments, the lysis buffer comprises a salt.In some embodiments, the lysis buffer comprises a detergent. In someembodiments, the detergent is SDS, Triton, or Tween. In someembodiments, the lysis buffer comprises a chemical which causes celllysis.

Any type of cellular nucleic acid can be transferred to the nucleic acidencoding. In some embodiments, the method comprises transferring one ormore cellular nucleic acids from the sample to the nucleic acidencoding. In some embodiments, the nucleic acids are mRNA. In someembodiments, the nucleic acids are mRNA that express a protein ofinterest. In some embodiments, the nucleic acids are genomic DNA. Insome embodiments, the nucleic acids are added as antibody-DNAconstructs. In some embodiments, the nucleic acids added are proximityligation products. In some embodiments, the nucleic acids added areproximity extension products. In some embodiments, a plurality ofdifferent cellular nucleic acids are attached to nucleic acid encodings.

In some embodiments, the nucleic acids transferred to the encodingcomprise a complementary sequence to a sequence on the encoding. Thismay allow for the ligation of the sample nucleic acid with the encodingnucleic acid via various methods. These methods include, withoutlimitation, annealing, ligating, chemically cross-linking, or amplifyingthe cellular contents on to the nucleic acid encoding the effector. Insome embodiments, the nucleic acid encodings comprise a sequencecomplementary to the nucleic acid of interest to be transferred to theencoding. This complementary sequence allows for the nucleic acids tohybridize with the encoding, which in turn allows for extension of theencoding with the cellular nucleic acid and vice versa.

In some embodiments, additional reagents are added to the encapsulationto facilitate the transfer of the nucleic acids to the encoding. In someembodiments, the additional reagents comprise an enzyme that facilitatesthe transfer of the nucleic acids. In some embodiments, the reagents fortransferring the nucleic acids to the encoding are added duringencapsulation step. In some embodiments, the reagents for transferringthe nucleic acids to the encoding are added during an incubation step.In some embodiments, the reagents for transferring the nucleic acids tothe encoding are added after an incubation step.

In some embodiments, the additional reagents to facilitate the transferof the nucleic acids comprise an enzyme. In some embodiments, the enzymeis a polymerase, a ligase, a restriction enzyme, or a recombinase. Insome embodiments, the enzyme is a polymerase. In some embodiments, theadditional reagents comprise a chemical cross-linking reagent. In someembodiments, the chemical cross-linking reagent is psoralen.

Adding Reagents to an Encapsulation

Methods and systems described herein may include adding one or morereagents to an encapsulation. In some embodiments, additional reagentscan be added during a screen to encapsulations by pico-injection. Insome embodiments, additional reagents are added by pico-injection. Insome embodiments, each encapsulation passing by a pico-injection sitereceive a pico-injection. In some embodiments, at least 80%, at least85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least99% of encapsulations passing a pico-injection site receivepico-injections. In some embodiments, at least 80% of encapsulationspassing a pico-injection site receive pico-injections. In someembodiments, at least 85% of encapsulations passing a pico-injectionsite receive pico-injections. In some embodiments, at least 90% ofencapsulations passing a pico-injection site receive pico-injections. Insome embodiments, at least 95% of encapsulations passing apico-injection site receive pico-injections. In some embodiments, atleast 97% of encapsulations passing a pico-injection site receivepico-injections. In some embodiments, at least 98% of encapsulationspassing a pico-injection site receive pico-injections. In someembodiments, at least 99% of encapsulations passing a pico-injectionsite receive pico-injections.

In some embodiments, pico-injections are performed at the same frequencyat which encapsulations pass by a pico-injection site. In someembodiments, pico-injections are performed at substantially the samefrequency at which encapsulations pass by a pico-injection site. In someembodiments, the frequency at which encapsulations pass by apico-injection site is determined by monitoring the encapsulations. Insome embodiments, the frequency at which encapsulations pass by apico-injection site is determined by monitoring the encapsulations inflow. In some embodiments, the encapsulations are monitored by takingimages in real time. In some embodiments, the encapsulations aremonitored with a detector.

In some embodiments, the pico-injections are conditional. Conditionalpico-injections may only occur after a certain condition is met. In someembodiments, a conditional pico-injection only occurs when a signal isdetected. In some embodiments, a reagent is injected by pico-injectionif a signal is detected. In some embodiments, a reagent is added to anencapsulation by pico-injections if a signal is detected. In someembodiments, the signal must be above a pre-determined threshold.

In some embodiments, a method for screening an encoded effectorcomprises providing an encapsulation comprising a sample and one or morescaffolds, wherein the scaffold comprises: an encoded effector bound tothe scaffold by a cleavable linker and a nucleic acid encoding theeffector; adding one or more reagents to the encapsulation throughpico-injection or by droplet merging; cleaving the cleavable linker torelease a pre-determined amount of the effector; detecting one or moresignals from the encapsulation, wherein the signal results from aninteraction between the encoded effector and the sample; and sorting theencapsulation based on the detection of the signal.

In some embodiments, one or more reagents added to an encapsulationcomprises one or more fluorophores, one or more antibodies, one or morechemical compounds, or any combination thereof.

Post-Sorting of Encapsulations

After a sorting step or barcoding step based on the detection of thesignal of interest, the results are deconvoluted in order to determinewhich effectors displayed the activity of interest against the targetsample. In some embodiments, the methods described herein comprise thestep of ascertaining which encodings are present in the samples sortedbased on the detection of the signal. In some embodiments wherein theencoding is a nucleic acid, the methods described herein furthercomprise the step of sequencing the encodings. In some embodiments, theencodings are sequenced by next generation sequencing. In someembodiments, the sequences are compared to a reference to ascertainwhich effectors displayed the activity of interest in the screen.

In some embodiments, sequencing the nucleic acid encoding comprisessequencing the encoding while the encoding is still attached to thescaffold. In some embodiments, sequencing the nucleic acid encodingcomprises cleaving the nucleic acid encoding from the scaffold. In someembodiments, sequencing the nucleic acid encoding comprises cleaving thenucleic acid encoding from the scaffold prior to sequencing. In someembodiments, cleaving the nucleic acid encoding from the scaffoldcomprises cleaving a cleavable linker with a cleaving reagent. In someembodiments, cleaving the nucleic acid encoding from the scaffoldcomprises cleaving a cleavable linker with electromagnetic radiation. Insome embodiments, any of the cleavable linkers and cleaving reagentsdescribed herein work for this purpose. In some embodiments, a nickingenzyme or a restriction enzyme can be used to cleave. In someembodiments enzymatic, chemical reagent, photocleavage can be used tocleave the encodings.

In some embodiments, the nucleic acid encoding comprises a sequencingprimer. The sequencing primer allows for facile amplification of thenucleic acid encoding. In some embodiments comprising a library ofencoded effectors, the sequencing primer is the same for each encoding.In some embodiments comprising a library of encoded effectors, thesequencing primer differs among the encodings. In some embodiments, thesequencing primer is upstream of the encoding. In some embodiments, thesequencing primer is downstream of the encoding.

In some embodiments, the methods provided herein are performed usingmicrofluidic devices. Microfluidic devices may perform the encapsulationsteps. Additionally, microfluidic devices may be equipped withpico-injectors and other components which allow for the methods providedherein to be performed. In some embodiments, pico-injectors are in placealong microfluidic channels defining a flow path through themicrofluidic device. In some embodiments, the pico-injectors arepositioned such that reagents are added at desired times whileperforming the methods provided herein.

In some embodiments, the methods and systems provided herein utilizelibraries of encoded effectors. Libraries of encoded effectors comprisea plurality of different effectors, each uniquely encoded by a knownencoding modality, such as those described above. Libraries may containany number of encoded effectors. In some embodiments, the librariescomprise at least 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹²,10¹³, 10¹⁴, 10¹⁵, or 10¹⁶ unique effectors. In some embodiments, thelibraries comprise at least about 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹,10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, or 10¹⁶ unique effectors.

In some embodiments, libraries of encoded effectors are linked toscaffolds. These scaffolds may be referred to as “scaffold encodedlibraries.” Scaffold encoded libraries comprise a plurality of encodedeffector molecules linked to the scaffold. The scaffold acts as a solidsupport and keeps the encoded effector molecules linked in space totheir encodings. In some embodiments, the libraries comprise at least10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵,or 10¹⁶ scaffolds. In some embodiments, the libraries comprise at leastabout 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴,10¹⁵, or 10¹⁶ scaffolds.

Any of the methods or systems described herein for a single encodedeffector may be utilized by a library of encoded effectors. In someembodiments, provided herein, is a method of screening a library ofencoded effectors, the method comprising using any of the methodspreviously described herein with a library of encoded effectors.

In some embodiments, libraries of encoded effectors comprise a pluralityof different encoded effectors. In some embodiments, libraries comprisemultiple copies of substantially identical effectors or scaffold encodedeffectors.

Microfluidic Devices

The methods and systems provided herein may be performed on amicrofluidic device. Device architecture and methods may be accomplishedin a variety of ways. An analyzer or sorter device according to thedisclosure comprises at least one analysis unit having an inlet regionin communication with a main channel at a droplet extrusion region(e.g., for introducing droplets of a sample into the main channel), adetection region within or coincident with all or a portion of the mainchannel or droplet extrusion region, and a detector associated with thedetection region. In certain embodiments the device may have two or moredroplet extrusion regions. For example, embodiments are provided inwhich the analysis unit has a first inlet region in communication withthe main channel at a first droplet extrusion region, a second inletregion in communication with the main channel at a second dropletextrusion region (for example, downstream from the first dropletextrusion region), and so forth.

In some embodiments, a microfluidic device described herein isconfigured for a droplet generation frequency of about 5 Hz to about 200Hz. In some embodiments, a microfluidic device described herein isconfigured for a throughput of about 5 Hz to about 15 Hz, about 5 Hz toabout 25 Hz, about 5 Hz to about 50 Hz, about 5 Hz to about 80 Hz, about5 Hz to about 100 Hz, about 5 Hz to about 150 Hz, about 5 Hz to about200 Hz, about 15 Hz to about 25 Hz, about 15 Hz to about 50 Hz, about 15Hz to about 80 Hz, about 15 Hz to about 100 Hz, about 15 Hz to about 150Hz, about 15 Hz to about 200 Hz, about 25 Hz to about 50 Hz, about 25 Hzto about 80 Hz, about 25 Hz to about 100 Hz, about 25 Hz to about 150Hz, about 25 Hz to about 200 Hz, about 50 Hz to about 80 Hz, about 50 Hzto about 100 Hz, about 50 Hz to about 150 Hz, about 50 Hz to about 200Hz, about 80 Hz to about 100 Hz, about 80 Hz to about 150 Hz, about 80Hz to about 200 Hz, about 100 Hz to about 150 Hz, about 100 Hz to about200 Hz, or about 150 Hz to about 200 Hz, including increments therein.In some embodiments, a microfluidic device described herein isconfigured for a droplet generation frequency of about 5 Hz, about 15Hz, about 25 Hz, about 50 Hz, about 80 Hz, about 100 Hz, about 150 Hz,or about 200 Hz. In some embodiments, a microfluidic device describedherein is configured for a droplet generation frequency of at leastabout 5 Hz, about 15 Hz, about 25 Hz, about 50 Hz, about 80 Hz, about100 Hz, or about 150 Hz. In some embodiments, a microfluidic devicedescribed herein is configured for a droplet generation frequency of atmost about 15 Hz, about 25 Hz, about 50 Hz, about 80 Hz, about 100 Hz,about 150 Hz, or about 200 Hz.

Sorter embodiments of the device also have a discrimination region orbranch point in communication with the main channel and with branchchannels, and a flow control responsive to the detector. There may be aplurality of detection regions and detectors, working independently ortogether, e.g., to analyze one or more properties of a sample orencapsulation. The branch channels may each lead to an outlet region andto a well or reservoir. There may also be a plurality of inlet regions,each of which introduces droplets of a different sample (e.g., of cells,of virions or of molecules such as molecules of an enzyme or asubstrate) into the main channel. Each of the one or more inlet regionsmay also communicate with a well or reservoir.

As each droplet passes into the detection region, it is examined for apredetermined characteristic or activity (i.e., using the detector) anda corresponding signal is produced, for example indicating that “yes”the characteristic or activity is present, or “no” it is not. The signalmay correspond to a characteristic qualitatively or quantitatively. Thatis, the amount of the signal can be measured and can correspond to thedegree to which a characteristic or activity is present. For example,the strength of the signal may indicate the size of a molecule, or thepotency or amount of an enzyme expressed by a cell, or a positive ornegative reaction such as binding or hybridization of one molecule toanother, a chemical reaction of a substrate catalyzed by an enzyme, orthe activation or inhibition of an enzyme, or any other type ofresponse. In response to the signal, data can be collected and/or a flowcontrol can be activated to divert a droplet into one branch channel oranother. Thus, samples within a droplet at a discrimination region canbe sorted into an appropriate branch channel according to a signalproduced by the corresponding examination at a detection region. In someembodiments, optical detection of molecular, cellular, viral, or othersample characteristics is used, for example directly or by use of areporter associated with a characteristic chosen for sorting. However,other detection techniques may also be employed.

A variety of channels for sample flow and mixing can be microfabricatedon a single chip and can be positioned at any location on the chip asthe detection and discrimination or sorting points, e.g., for kineticstudies. A plurality of analysis units of the disclosure may be combinedin one device. Microfabrication applied according to the disclosureeliminates the dead time occurring in conventional gel electrophoresisor flow cytometric kinetic studies, and achieves a bettertime-resolution. Furthermore, linear arrays of channels on a singlechip, i.e., a multiplex system, can simultaneously detect and sort asample by using an array of photo multiplier tubes (PMT) for parallelanalysis of different channels. This arrangement can be used to improvethroughput or for successive sample enrichment, and can be adapted toprovide a very high throughput to the microfluidic devices that exceedsthe capacity permitted by conventional flow sorters. Circulation systemscan be used in cooperation with these and other features of thedisclosure. Microfluidic pumps and valves are one way of controllingfluid and sample flow. See, for example, U.S. patent application Ser.No. 60/186,856.

Microfabrication permits other technologies to be integrated or combinedwith flow cytometry on a single chip, such as PCR, moving cells usingoptical tweezer/cell trapping, transformation of cells byelectroporation, μTAS, and DNA hybridization. Detectors and/or lightfilters that are used to detect viral (or cell) characteristics of thereporters can also be fabricated directly on the chip.

A device of the disclosure can be microfabricated with a sample solutionreservoir or well at the inlet region, which is typically in fluidcommunication with an inlet channel. A reservoir may facilitateintroduction of molecules or cells into the device and into the sampleinlet channel of each analysis unit. An inlet region may have an openingsuch as in the floor of the microfabricated chip, to permit entry of thesample into the device. The inlet region may also contain a connectoradapted to receive a suitable piece of tubing, such as liquidchromatography or HPLC tubing, through which a sample may be supplied.Such an arrangement facilitates introducing the sample solution underpositive pressure in order to achieve a desired pressure at the dropletextrusion region.

A device of the disclosure may have an additional inlet region, indirect communication with the main channel at a location upstream of thedroplet extrusion region, through which a pressurized stream or “flow”of a fluid is introduced into the main channel. In some embodiments,this fluid is one which is not miscible with the solvent or fluid of thesample. For example, in some embodiments, the fluid is a non-polarsolvent, such as decane (e.g., tetradecane or hexadecane), and thesample (e.g., of cells, virions or molecules) is dissolved or suspendedin an aqueous solution so that aqueous droplets of the sample areintroduced into the pressurized stream of non-polar solvent at thedroplet extrusion region.

Substrate and flow channels may be accomplished in a variety of ways. Atypical analysis unit of the disclosure comprises a main inlet that ispart of and feeds or communicates directly with a main channel, alongwith one or more sample inlets in communication with the main channel ata droplet extrusion region situated downstream from the main inlet (eachdifferent sample inlet may communicate with the main channel at adifferent droplet extrusion region). The droplet extrusion regiongenerally comprises a junction between the sample inlet and the mainchannel such that a pressurized solution of a sample (i.e., a fluidcontaining a sample such as cells, virions or molecules) is introducedto the main channel in droplets. In some embodiment, the sample inletintersects the main channel such that the pressurized sample solution isintroduced into the main channel at an angle perpendicular to a streamof fluid passing through the main channel. For example, in someembodiments, the sample inlet and main channel intercept at a T-shapedjunction; i.e., such that the sample inlet is perpendicular (90 degrees)to the main channel. However, the sample inlet may intercept the mainchannel at any angle, and need not introduce the sample fluid to themain channel at an angle that is perpendicular to that flow. In someembodiments the angle between intersecting channels is in the range offrom about 60 to about 120 degrees. Particular exemplary angles are 45,60, 90, and 120 degrees. In some embodiments, the angle between theintersecting channels is in the range of about 5 to about 60 degrees. Insome embodiments, the angle between the intersecting channels is in therange of about 5 to about 60 degrees. In some embodiments, the anglebetween the intersecting channels is in the range of about 5 to about10, about 5 to about 15, about 5 to about 20, about 5 to about 25, about5 to about 30, about 5 to about 40, about 5 to about 50, about 5 toabout 60, about 10 to about 15, about 10 to about 20, about 10 to about25, about 10 to about 30, about 10 to about 40, about 10 to about 50,about 10 to about 60, about 15 to about 20, about 15 to about 25, about15 to about 30, about 15 to about 40, about 15 to about 50, about 15 toabout 60, about 20 to about 25, about 20 to about 30, about 20 to about40, about 20 to about 50, about 20 to about 60, about 25 to about 30,about 25 to about 40, about 25 to about 50, about 25 to about 60, about30 to about 40, about 30 to about 50, about 30 to about 60, about 40 toabout 50, about 40 to about 60, or about 50 to about 60 degrees. In someembodiments, the angle between the intersecting channels is in the rangeof about 5, about 10, about 15, about 20, about 25, about 30, about 40,about 50, or about 60 degrees. In some embodiments, the angle betweenthe intersecting channels is in the range of at least about 5, about 10,about 15, about 20, about 25, about 30, about 40, or about 50. In someembodiments, the angle between the intersecting channels is in the rangeof at most about 10, about 15, about 20, about 25, about 30, about 40,about 50, or about 60 degrees. In some embodiments, the angle betweenthe intersecting channels is in the range of about 120 to about 175degrees. In some embodiments, the angle between the intersectingchannels is in the range of about 120 to about 130, about 120 to about140, about 120 to about 150, about 120 to about 160, about 120 to about170, about 120 to about 175, about 130 to about 140, about 130 to about150, about 130 to about 160, about 130 to about 170, about 130 to about175, about 140 to about 150, about 140 to about 160, about 140 to about170, about 140 to about 175, about 150 to about 160, about 150 to about170, about 150 to about 175, about 160 to about 170, about 160 to about175, or about 170 to about 175 degrees. In some embodiments, the anglebetween the intersecting channels is in the range of about 120, about130, about 140, about 150, about 160, about 170, or about 175 degrees.In some embodiments, the angle between the intersecting channels is inthe range of at least about 120, about 130, about 140, about 150, about160, or about 170 degrees. In some embodiments, the angle between theintersecting channels is in the range of at most about 130, about 140,about 150, about 160, about 170, or about 175 degrees.

The droplet extrusion or droplet formation region may also comprise twomicrofluidic channels carrying immiscible carrier fluid that areintroduced on opposite sides of a main microfluidic channel. In someembodiments, the two microfluidic channels are substantially collinear.In some embodiments, such a junction resembles and X-shape. In someembodiments, the main microfluidic channel contains the sample or assayfluid.

The main channel in turn communicates with two or more branch channelsat another junction or “branch point”, forming, for example, a T-shapeor a Y-shape. Other shapes and channel geometries may be used asdesired. In sorting embodiments, the region at or surrounding thejunction can also be referred to as a discrimination region or a sortingregion. Precise boundaries for the discrimination region are notrequired, but are preferred.

A detection region may be within, communicating or coincident with aportion of the main channel at or downstream of the droplet extrusionregion and, in sorting embodiments, at or upstream of the discriminationregion or branch point. Precise boundaries for the detection region arenot required, but are preferred. The discrimination region may belocated immediately downstream of the detection region or it may beseparated by a suitable distance consistent with the size of themolecules, the channel dimensions and the detection system. It will beappreciated that the channels may have any suitable shape orcross-section (for example, tubular or grooved), and can be arranged inany suitable manner so long as flow can be directed from inlet to outletand from one channel into another.

The channels of the disclosure may be microfabricated, for example byetching a silicon chip using conventional photolithography techniques,or using a micromachining technology called “soft lithography”. Theseand other microfabrication methods may be used to provide inexpensiveminiaturized devices, and in the case of soft lithography, can providerobust devices having beneficial properties such as improvedflexibility, stability, and mechanical strength. When optical detectionis employed, the devices provided herein may also provide minimal lightscatter from molecule or cell (including virion) suspension and chambermaterial. In some embodiments, devices provided herein are relativelyinexpensive and easy to set up. They can also be disposable, whichgreatly relieves many of the concerns of gel electrophoresis (formolecules), and of sterilization and permanent adsorption of particlesinto the flow chambers and channels of conventional FACS machines (forcells, virions and other particle suspensions).

A microfabricated device of the disclosure may be fabricated from asilicon microchip or silicon elastomer. In some embodiments, thedimensions of the chip are those of typical microchips, ranging betweenabout 0.5 cm to about 5 cm per side and about 1 micron to about 1 cm inthickness. The device may contain at least one analysis unit having amain channel with a droplet extrusion region and a coincident detectionregion. The device may also contain at least one inlet region (which maycontain an inlet channel) and one or more outlet regions (which may havefluid communication with a branch channel in each region). In a sortingembodiment, at least one detection region cooperates with at least onediscrimination region to divert flow via a detector-originated signal.It shall be appreciated that the “regions” and “channels” are in fluidcommunication with each other and therefore may overlap; i.e., there maybe no clear boundary where a region or channel begins or ends. Amicrofabricated device can be transparent and can be covered with amaterial having transparent properties, such as a glass coverslip, topermit detection of a reporter, for example, by an optical device suchas an optical microscope.

The dimensions of the detection region are influenced by the nature ofthe sample under study and, in particular, by the size of the moleculesor cells (including virions) under study. For example, viruses can havea diameter from about 20 nm to about 500 nm, although some extremelylarge viruses may reach lengths of about 2000 nm (i.e., as large orlarger than some bacterial cells). By contrast, biological cells aretypically many times larger. For example, mammalian cells can have adiameter of about 1 to 50 microns, more typically 10 to 30 microns,although some mammalian cells (e.g., fat cells) can be larger than 120microns. Plant cells are generally 10 to 100 microns.

Detection regions used for detecting molecules and cells (includingvirions) have a cross-sectional area large enough to allow a desiredmolecule to pass through without being substantially slowed downrelative to the flow carrying it. To avoid “bottlenecks” and/orturbulence, and promote single-file flow, the channel dimensions,particularly in the detection region, should generally be at least abouttwice, or at least about five times as large per side or in diameter asthe diameter of the largest molecule, cell or droplet that will bepassing through it.

For particles (e.g., cells, including virions) or molecules that are inencapsulations (i.e., deposited by the droplet extrusion region) withinthe flow of the main channel, the channels of the device may be rounded,with a diameter between about 2 and 100 microns. In some embodiments,the round channels of the device are about 60 microns in diameter orabout 30 microns at the crossflow area or droplet extrusion region. Thisgeometry facilitates an orderly flow of droplets in the channels.Similarly, the volume of the detection region in an analysis device maybe in the range of between about 10 femtoliters (fl) and 5000 fl, about40 or 50 fl to about 1000 or 2000 fl, or on the order of about 200 fl.In some embodiments, the channels of the device, and particularly thechannels of the inlet connecting to a droplet extrusion region, arebetween about 2 and 50 microns, or about 30 microns.

In one embodiment, droplets at these dimensions tend to conform to thesize and shape of the channels, while maintaining their respectivevolumes. Thus, as droplets move from a wider channel to a narrowerchannel they become longer and thinner, and vice versa. In someembodiments, droplets are at least about four times as long as they arewide. This droplet configuration, which can be envisioned as a lozengeshape, flows smoothly and well through the channels. Longer droplets,produced in narrower channels, provides a higher shear, meaning thatdroplets can more easily be sheared or broken off from a flow, i.e.using less force. Droplets may also tend to adhere to channel surfaces,which can slow or block the flow, or produce turbulence. Dropletadherence is overcome when the droplet is massive enough in relation tothe channel size to break free. Thus, droplets of varying size, ifpresent, may combine to form uniform droplets having a so-calledcritical mass or volume that results in smooth or laminar droplet flow.Droplets that are longer than they are wide, for example about fourtimes longer than they are wide, generally have the ability to overcomechannel adherence and move freely through the microfluidic device. Thus,in an exemplary embodiment with 60 micron channels, a typicalfree-flowing droplet is about 60 microns wide and 240 microns long.Droplet dimensions and flow characteristics can be influenced asdesired, in part by changing the channel dimensions, e.g. the channelwidth.

In some embodiments, the devices provided herein generate round,monodisperse droplets. In some embodiments, the droplets have a diameterthat is smaller than the diameter of the microchannel; i.e., less than60 μm. Monodisperse droplets may be particularly preferable, e.g., inhigh throughput devices and other embodiments where it is desirable togenerate droplets at high frequency.

To prevent sample (e.g., cells, virions and other particles ormolecules) or other material from adhering to the sides of the channels,the channels (and coverslip, if used) may have a coating which minimizesadhesion. Such a coating may be intrinsic to the material from which thedevice is manufactured, or it may be applied after the structuralaspects of the channels have been microfabricated. “TEFLON” is anexample of a coating that has suitable surface properties.Alternatively, the channels may be coated with a surfactant.

Non-limiting examples of surfactants that may be used include, but arenot limited to, surfactants such as sorbitan-based carboxylic acidesters (e.g., the “Span” surfactants, Fluka Chemika), including sorbitanmonolaurate (Span20), sorbitan monopalmitate (Spa n 40), sorbitanmonostearate (Span60) and sorbitan monooleate (Span80). Othernon-limiting examples of non-ionic surfactants which may be used includepolyoxyethylenated alkylphenols (for example, nonyl-, p-dodecyl-, anddinonylphenols), polyoxyethylenated straight chain alcohols,polyoxyethylenated polyoxypropylene glycols, polyoxyethylenatedmercaptans, long chain carboxylic acid esters (for example, glyceryl andpolyglyceryl esters of natural fatty acids, propylene glycol, sorbitol,polyoxyethylenated sorbitol esters, polyoxyethylene glycol esters, etc.)and alkanolamines (e.g., diethanolamine-fatty acid condensates andisopropanolamine-fatty acid condensates). In addition, ionic surfactantssuch as sodium dodecyl sulfate (SDS) may also be used.

A silicon substrate containing the microfabricated flow channels andother components may be covered and sealed, including with a transparentcover, e.g., thin glass or quartz, although other clear or opaque covermaterials may be used. When external radiation sources or detectors areemployed, the detection region may be covered with a clear covermaterial to allow optical access to the cells. For example, anodicbonding to a “PYREX” cover slip can be accomplished by washing bothcomponents in an aqueous H₂SO₄/H₂O₂ bath, rinsing in water, and then,for example, heating to about 350° C. while applying a voltage of 450V.

Switching and flow control can be accomplished in a variety of ways.Some embodiments of the disclosure use pressure drive flow control,e.g., utilizing valves and pumps, to manipulate the flow of cellsvirions, particles, molecules, enzymes or reagents in one or moredirections and/or into one or more channels of a microfluidic device.However, other methods may also be used, alone or in combination withpumps and valves, such as electro-osmotic flow control, electrophoresisand dielectrophoresis. In certain embodiments of the disclosure, theflow moves in one “forward” direction, e.g. from the main inlet regionthrough the main and branch channels to an outlet region. In otherembodiments the direction of flow is reversible. Application of thesetechniques according to the disclosure provides more rapid and accuratedevices and methods for analysis or sorting, for example, because thesorting occurs at or in a discrimination region that can be placed at orimmediately after a detection region. This provides a shorter distancefor molecules or cells to travel, they can move more rapidly and withless turbulence, and can more readily be moved, examined, and sorted insingle file, i.e., one at a time. In a reversible embodiment, potentialsorting errors can be avoided, for example by reversing and slowing theflow to re-read or resort a molecule, cell or virion (or pluralitiesthereof) before irretrievably committing the cell or cells to aparticular branch channel.

Without being bound by any theory, electro-osmosis is believed toproduce motion in a stream containing ions, e.g. a liquid such as abuffer, by application of a voltage differential or charge gradientbetween two or more electrodes. Neutral (uncharged) molecules or cells(including virions) can be carried by the stream. Electro-osmosis isparticularly suitable for rapidly changing the course, direction orspeed of flow. Electrophoresis is believed to produce movement ofcharged objects in a fluid toward one or more electrodes of oppositecharge, and away from one on or more electrodes of like charge. Inembodiments of the disclosure where an aqueous phase is combined with anoil phase, aqueous droplet encapsulations are encapsulated or separatedfrom each other by oil. In some embodiments, the oil phase is not anelectrical conductor and may insulate the encapsulations from theelectro-osmotic field. In these embodiment, electro-osmosis may be usedto drive the flow of encapsulations if the oil is modified to carry orreact to an electrical field, or if the oil is substituted for anotherphase that is immiscible in water but which does not insulate the waterphase from electrical fields.

Dielectrophoresis produces dielectric objects, which have no net charge,but have regions that are positively or negatively charged in relationto each other. Alternating, non-homogeneous electric fields in thepresence of encapsulations, including droplets, and/or particles, suchas cells or virions, cause the encapsulations and/or particles to becomeelectrically polarized and thus to experience dielectrophoretic forces.Depending on the dielectric polarizability of the particles and thesuspending medium, dielectric particles will move either toward theregions of high field strength or low field strength. For example, thepolarizability of living cells and virions depends on their composition,morphology, and phenotype and is highly dependent on the frequency ofthe applied electrical field. Thus, cells and virions of different typesand in different physiological states generally possess distinctlydifferent dielectric properties, which may provide a basis for cellseparation, e.g., by differential dielectrophoretic forces. Likewise,the polarizability of encapsulations, including droplets, also dependsupon their size, shape and composition. For example, droplets thatcontain salts can be polarized. Individual manipulation of singleencapsulations requires field differences (inhomogeneities) withdimensions close to the encapsulations.

Manipulation is also dependent on permittivity (a dielectric property)of the encapsulations and/or particles with the suspending medium. Thus,polymer particles, living cells and virions show negativedielectrophoresis at high-field frequencies in water. For example,dielectrophoretic forces experienced by a latex sphere in a 0.5 MV/mfield (10V for a 20 micron electrode gap) in water are predicted to beabout 0.2 piconewtons (pN) for a 3.4 micron latex sphere to 15 pN for a15 micron latex sphere. These values are mostly greater than thehydrodynamic forces experienced by the sphere in a stream (about 0.3 pNfor a 3.4 micron sphere and 1.5 pN for a 15 micron sphere). Therefore,manipulation of individual cells or particles can be accomplished in astreaming fluid, such as in a cell sorter device, usingdielectrophoresis. Using conventional semiconductor technologies,electrodes can be microfabricated onto a substrate to control the forcefields in a microfabricated sorting device of the disclosure.Dielectrophoresis is particularly suitable for moving objects that areelectrical conductors. AC current may be used to prevent permanentalignment of ions. Megahertz frequencies are suitable to provide a netalignment, attractive force, and motion over relatively long distances.

Radiation pressure can also be used in the disclosure to deflect andmove objects, e.g. encapsulations, droplets, and particles (molecules,cells, virions, etc.) contained therein, with focused beams of lightsuch as lasers. Flow can also be obtained and controlled by providing apressure differential or gradient between one or more channels of adevice or in a method of the disclosure.

In some embodiments, molecules, cells or virions (or droplets containingmolecules, cells or virions) can be moved by direct mechanicalswitching, e.g., with on-off valves or by squeezing the channels.Pressure control may also be used, for example, by raising or loweringan output well to change the pressure inside the channels on the chip.Different switching and flow control mechanisms can be combined on onechip or in one device and can work independently or together as desired.

Detection and discrimination for sorting can be accomplished in avariety of ways. The detector can be any device or method forinterrogating a molecule, a cell or a virion as it passes through thedetection region. Typically, molecules, cells or virions (or dropletscontaining such particles) are to be analyzed or sorted according to apredetermined characteristic that is directly or indirectly detectable,and the detector is selected or adapted to detect that characteristic.One detector is an optical detector, such as a microscope, which may becoupled with a computer and/or other image processing or enhancementdevices to process images or information produced by the microscopeusing known techniques. For example, molecules can be analyzed and/orsorted by size or molecular weight. Enzymes can be analyzed and/orsorted by the extent to which they catalyze chemical reaction of asubstrate (conversely, substrate can be analyzed and/or sorted by thelevel of chemical reactivity catalyzed by an enzyme). Cells and virionscan be sorted according to whether they contain or produce a particularprotein, by using an optical detector to examine each cell or virion foran optical indication of the presence or amount of that protein. Theprotein may itself be detectable, for example by a characteristicfluorescence, or it may be labeled or associated with a reporter thatproduces a detectable signal when the desired protein is present, or ispresent in at least a threshold amount. There is no limit to the kind ornumber of characteristics that can be identified or measured using thetechniques of the disclosure, which include without limitation surfacecharacteristics of the cell or virion and intracellular characteristics,provided only that the characteristic or characteristics of interest forsorting can be sufficiently identified and detected or measured todistinguish cells having the desired characteristic(s) from those whichdo not. For example, any label or reporter as described herein can beused as the basis for analyzing and/or sorting molecules or cells(including virions), i.e. detecting molecules or cells to be collected.

In some embodiments, the samples (or encapsulations containing them) areanalyzed and/or separated based on the intensity of a signal from anoptically-detectable reporter bound to or associated with them as theypass through a detection window or “detection region” in the device. Insome embodiments, the samples are analyzed and/or separated based on theintensity of a signal from a detectable reporter. Molecules or cells orvirions having an amount or level of the reporter at a selectedthreshold or within a selected range are diverted into a predeterminedoutlet or branch channel of the device. The reporter signal may becollected by a microscope and measured by a photo multiplier tube (PMT).A computer digitizes the PMT signal and controls the flow via valveaction or electro-osmotic potentials. Alternatively, the signal can berecorded or quantified as a measure of the reporter and/or itscorresponding characteristic or marker, e.g., for the purpose ofevaluation and without necessarily proceeding to sort the molecules orcells.

In one embodiment, the chip is mounted on an inverted opticalmicroscope. Fluorescence produced by a reporter is excited using a laserbeam focused on molecules (e.g., DNA, protein, enzyme or substrate) orcells passing through a detection region. Fluorescent reporters include,e.g., rhodamine, fluorescein, Texas red, Cy 3, Cy 5, phycobiliprotein,green fluorescent protein (GFP), YOYO-1 and PicoGreen, to name a few. Inmolecular fingerprinting applications, the reporter labels areoptionally fluorescently labeled single nucleotides, such asfluorescein-dNTP, rhodamine-dNTP, Cy3-dNTP, etc.; where dNTP representsdATP, dTTP, dUTP or dCTP. The reporter can also be chemically-modifiedsingle nucleotides, such as biotin-dNTP. In other embodiments, thereporter can be fluorescently or chemically labeled amino acids orantibodies (which bind to a particular antigen, or fragment thereof,when expressed or displayed by a cell or virus).

Thus, in one aspect of the disclosure, the device can analyze and/orsort cells or virions based on the level of expression of selected cellmarkers, such as cell surface markers, which have a detectable reporterbound thereto, in a manner similar to that currently employed usingfluorescence-activated cell sorting (FACS) machines. Proteins or othercharacteristics within a cell, and which do not necessarily appear onthe cell surface, can also be identified and used as a basis forsorting. In another aspect of the disclosure, the device can determinethe size or molecular weight of molecules such as polynucleotides orpolypeptides (including enzymes and other proteins) or fragments thereofpassing through the detection region. Alternatively, the device candetermine the presence or degree of some other characteristic indicatedby a reporter. If desired, the cells, virions or molecules can be sortedbased on this analysis. The sorted cells, virions or molecules can becollected from the outlet channels and used as needed.

To detect a reporter or determine whether a molecule, cell or virion hasa desired characteristic, the detection region may include an apparatusfor stimulating a reporter for that characteristic to emit measurablelight energy, e.g., a light source such as a laser, laser diode,high-intensity lamp, (e.g., mercury lamp), and the like. In embodimentswhere a lamp is used, the channels may be shielded from light in allregions except the detection region. In embodiments where a laser isused, the laser can be set to scan across a set of detection regionsfrom different analysis units. In addition, laser diodes may bemicrofabricated into the same chip that contains the analysis units.Alternatively, laser diodes may be incorporated into a second chip(i.e., a laser diode chip) that is placed adjacent to themicrofabricated analysis or sorter chip such that the laser light fromthe diodes shines on the detection region(s).

In some embodiments, an integrated semiconductor laser and/or anintegrated photodiode detector are included on the silicon wafer in thevicinity of the detection region. This design provides the advantages ofcompactness and a shorter optical path for exciting and/or emittedradiation, thus minimizing distortion.

Sorting schemes can be accomplished in a variety of ways. According tothe disclosure, molecules (such as DNA, protein, enzyme or substrate) orparticles (i.e., cells, including virions) are sorted dynamically in aflow stream of microscopic dimensions based on the detection ormeasurement of a characteristic, marker or reporter that is associatedwith the molecules or particles. More specifically, encapsulations of asolution (for example an aqueous solution or buffer), containing asample of molecules, cells or virions, are introduced through a dropletextrusion region into a stream of fluid (for example, a non-polar fluidsuch as decane or other oil) in the main channel. The individual dropletencapsulations are then analyzed and/or sorted in the flow stream,thereby sorting the molecules, cells or virions contained within thedroplets.

The flow stream in the main channel is typically, but not necessarilycontinuous and may be stopped and started, reversed or changed in speed.Prior to sorting, a liquid that does not contain samples molecules,cells or virions can be introduced into a sample inlet region (such asan inlet well or channel) and directed through the droplet extrusionregion, e.g., by capillary action, to hydrate and prepare the device foruse. Likewise, buffer or oil can also be introduced into a main inletregion that communicates directly with the main channel to purge thedevice (e.g., or “dead” air) and prepare it for use. If desired, thepressure can be adjusted or equalized, for example, by adding buffer oroil to an outlet region.

The pressure at the droplet extrusion region can also be regulated byadjusting the pressure on the main and sample inlets, for example, withpressurized syringes feeding into those inlets. By controlling thepressure difference between the oil and water sources at the dropletextrusion region, the size and periodicity of the droplets generated maybe regulated. Alternatively, a valve may be placed at or coincident toeither the droplet extrusion region or the sample inlet connectedthereto to control the flow of solution into the droplet extrusionregion, thereby controlling the size and periodicity of the droplets.Periodicity and droplet volume may also depend on channel diameter, theviscosity of the fluids, and shear pressure.

The droplet forming liquid is typically an aqueous buffer solution, suchas ultrapure water (e.g., 18 mega-ohm resistivity, obtained, for exampleby column chromatography), 10 mM Tris HCl and 1 mM EDTA (TE) buffer,phosphate buffer saline (PBS) or acetate buffer. Any liquid or bufferthat is physiologically compatible with the population of molecules,cells or virions to be analyzed and/or sorted can be used. The fluidpassing through the main channel and in which the droplets are formed ispreferably one that is not miscible with the droplet forming fluid. Insome embodiments, the fluid passing through the main channel is anon-polar solvent, for example decane (e.g., tetradecane or hexadecane)or another oil.

The fluids used in the disclosure may contain additives, such as agentswhich reduce surface tensions (surfactants). Exemplary surfactantsinclude Tween, Span, fluorinated oils, and other agents that are solublein oil relative to water. Surfactants may aid in controlling oroptimizing droplet size, flow and uniformity, for example by reducingthe shear force needed to extrude or inject droplets into anintersecting channel. This may affect droplet volume and periodicity, orthe rate or frequency at which droplets break off into an intersectingchannel.

Channels of the disclosure may be formed from silicon elastomer (e.g.RTV), urethane compositions, of from silicon-urethane composites such asthose available from Polymer Technology Group (Berkeley, Calif.), e.g.PurSil™ and CarboSil™. The channels may also be coated with additives oragents, such as surfactants, TEFLON, or fluorinated oils such asoctadecafluoroctane (98%, Aldrich) or fluorononane. TEFLON isparticularly suitable for silicon elastomer (RTV) channels, which arehydrophobic and advantageously do not absorb water, but they may tend toswell when exposed to an oil phase. Swelling may alter channeldimensions and shape, and may even close off channels, or may affect theintegrity of the chip, for example by stressing the seal between theelastomer and a coverslip. Urethane substrates do not tend to swell inoil but are hydrophilic, they may undesirably absorb water, and tend touse higher operating pressures. Hydrophobic coatings may be used toreduce or eliminate water absorption. Absorption or swelling issues mayalso be addressed by altering or optimizing pressure or dropletfrequency (e.g. increasing periodicity to reduce absorption).RTV-urethane hybrids may be used to combine the hydrophobic propertiesof silicon with the hydrophilic properties of urethane.

Embodiments of the disclosure are also provided in which there are twoor more droplet formation regions introducing droplets of samples intothe main channel. For example, a first droplet extrusion region mayintroduce droplets of a first sample into a flow of fluid (e.g., oil) inthe main channel and a second droplet extrusion region may introducedroplets of a second sample into the flow of fluid in main channel, andso forth. Optionally, the second droplet extrusion region is downstreamfrom the first droplet extrusion region (e.g., about 30 μm). In oneembodiment, the fluids introduced into the two or more different dropletextrusion regions comprise the same fluid or the same type of fluid(e.g., different aqueous solutions). For example, in one embodimentdroplets of an aqueous solution containing an enzyme are introduced intothe main channel at the first droplet extrusion region and droplets ofaqueous solution containing a substrate for the enzyme are introducedinto the main channel at the second droplet extrusion region. Theintroduction of droplets through the different extrusion regions may becontrolled, e.g., so that the droplets combine (allowing, for example,the enzyme to catalyze a chemical reaction of the substrate).Alternatively, the droplets introduced at the different dropletextrusion regions may be droplets of different fluids which may becompatible or incompatible. For example, the different droplets may bedifferent aqueous solutions, or droplets introduced at a first dropletextrusion region may be droplets of one fluid (e.g., an aqueoussolution) whereas droplets introduced at a second droplet extrusionregion may be another fluid (e.g., alcohol or oil).

The concentration (i.e., number) of scaffolds, molecules, cells orvirions in a droplet can influence sorting efficiently and therefore maybe optimized. In particular, the sample concentration should be diluteenough that most of the droplets contain no more than a singlesscaffold, molecule, cell or virion, with only a small statistical chancethat a droplet will contain two or more molecules, cells or virions. Insome embodiments, the sample concentration should be such that a singlecell is encapsulated with a single scaffold. This is to ensure that forthe large majority of measurements, the level of reporter measured ineach droplet as it passes through the detection region corresponds to asingle molecule, cell or virion and not to two or more molecules, cellsor virions. Additionally, ensuring that a single cell or virion isencapsulated with only a single encoded effector scaffold ensures thatpositive “hits” are correctly correlated with the correct effectors.

The parameters which govern this relationship are the volume of thedroplets and the concentration of molecules, cells or virions in thesample solution. The probability that a droplet will contain two or morescaffolds, molecules, cells, or virions (P≤2) can be expressed as

P≤2=1−{1+[virion]×V}×e−[virion]×V

where “[virion]” is the concentration of molecules, cells or virions inunits of number of molecules, cells or virions per cubic micron (μm3),and V is the volume of the droplet in units of μm3.

It will be appreciated that P≤2 can be minimized by decreasing theconcentration of scaffolds, molecules, cells or virions in the samplesolution. However, decreasing the concentration of molecules, cells orvirions in the sample solution also results in an increased volume ofsolution processed through the device and can result in longer runtimes. Accordingly, it is desirable to minimize to presence of multiplemolecules, cells or virions in the droplets (thereby increasing theaccuracy of the sorting) and to reduce the volume of sample, therebypermitting a sorted sample in a reasonable time in a reasonable volumecontaining an acceptable concentration of molecules, cells or virions.

The maximum tolerable P≤2 depends on the desired “purity” of the sortedsample. The “purity” in this case refers to the fraction of sortedmolecules, cells or virions that possess a desired characteristic (e.g.,display a particular antigen, are in a specified size range or are aparticular type of molecule, cell or virion). The purity of the sortedsample is inversely proportional to P≤2. For example, in applicationswhere high purity is not needed or desired a relatively high P≤2 (e.g.,P≤2=0.2) may be acceptable. For most applications, maintaining P≤2 at orbelow about 0.1, or at or below about 0.01, provides satisfactoryresults.

A sample solution containing a mixture or population of molecule, cellsor virions in a suitable carrier fluid (such as a liquid or bufferdescribed above) is supplied to the sample inlet region, and droplets ofthe sample solution are introduced, at the droplet extrusion region,into the flow passing through the main channel. The force and directionof flow can be controlled by any desired method for controlling flow,for example, by a pressure differential, by valve action or byelectro-osmotic flow (e.g., produced by electrodes at inlet and outletchannels). This permits the movement of the cells into one or moredesired branch channels or outlet regions.

A “forward” sorting algorithm, according to the disclosure, includesembodiments where droplets from a droplet extrusion region flow throughthe device to a predetermined branch or outlet channel (which can becalled a “waste channel”), until the level of measurable reporter of amolecule, cell or virion within a droplet is above a pre-set threshold.At that time, the flow is diverted to deliver the droplet (and thescaffold, molecule, cell, and/or virion contained therein) to anotherchannel. For example, in an electro-osmotic embodiment, where switchingis virtually instantaneous and throughput is limited by the highestvoltage, the voltages are temporarily changed to divert the chosendroplet to another predetermined outlet channel (which can be called a“collection channel”). Sorting, including synchronizing detection of areporter and diversion of the flow, can be controlled by various methodsincluding computer or microprocessor control. Different algorithms forsorting in the microfluidic device can be implemented by differentcomputer programs, such as programs used in conventional FACS devices.For example, a programmable card can be used to control switching, suchas a Lab PC 1200 Card, available from National Instruments, Austin, Tex.Algorithms as sorting procedures can be programmed using C++, LAB VIEW,or any suitable software.

A “reversible” sorting algorithm can be used in place of a “forward”mode, for example in embodiments where switching speed may be limited.For example, a pressure-switched scheme can be used instead ofelectro-osmotic flow and does not require high voltages and may be morerobust for longer runs. However, mechanical constraints may cause thefluid switching speed to become rate-limiting. In a pressure-switchedscheme the flow is stopped when a molecule or cell or virion of interestis detected within a droplet. By the time the flow stops, the dropletcontaining the molecule, cell or virion may be past the junction orbranch point and be part of the way down the waste channel. In thissituation, a reversible embodiment can be used. The system can be runbackwards at a slower (switchable) speed (e.g., from waste to inlet),and the droplet is then switched to a different branch or collectionchannel. At that point, a potentially mis-sorted droplet (and themolecule, cell or virion therein) is “saved”, and the device can againbe run at high speed in the forward direction. This “reversible” sortingmethod is not possible with standard FACS machines. FACS machines mostlysort aerosol droplets which cannot be reversed back to the chamber, inorder to be redirected. The aerosol droplet sorters are virtuallyirreversible. Reversible sorting is particularly useful for identifyingmolecules, cells or virions that are rare (e.g., in molecular evolutionand cancer cytological identification) or few in number, which may bemisdirected due to a margin of error inherent to any fluidic device. Thereversible nature of the device of the disclosure permits a reduction inthis possible error.

In addition, a “reversible” sorting method permits multiple time coursemeasurements of a molecule, cell or virion contained within a singledroplet. This allows for observations or measurements of the samemolecule, cell or virion at different times, because the flow reversesthe cell back into the detection window again before redirecting thecell into a different channel. Thus, measurements can be compared orconfirmed, and changes in properties over time can be examined, forexample in kinetic studies.

When trying to separate scaffolds, molecules, cells or virions in asample at a very low ratio to the total number of scaffolds, molecules,cells or virions, a sorting algorithm can be implemented that is notlimited by the intrinsic switching speed of the device. Consequently,the droplets flow at the highest possible static (non-switching) speedfrom the inlet channel to the waste channel. Unwanted droplets (i.e.,containing unwanted molecules, cells or virions) can be directed intothe waste channel at the highest speed possible, and when a dropletcontaining a desired molecule, cell or virion is detected, the flow canbe slowed down and then reversed, to direct the droplet back into thedetection region, from where it can be redirected (i.e. to accomplishefficient switching). Hence the droplets (and the molecules, cells orvirions contained therein) can flow at the highest possible staticspeed.

Provided herein are methods for controlling for variables such astemperature, pH and concentration. This may be accomplished byconverging two aqueous streams to form droplets, where, for example, thefirst aqueous stream would contain 2× the concentration of component “A”desired in the droplet and the second aqueous stream would contain 2×the concentration of component “B” desired in the droplet, thus when thestreams merge they would form a 1× solution of both “A” and “B”.Different ratios of aqueous streams converging with differentconcentrations of reagents may also be sued to reach desired finalconcentrations of samples, scaffolds, and/or reagents. Theconcentrations in droplets are controlled by knowing what theconcentrations are of components in each aqueous stream. This conceptcan be applied to pH, salt, concentration, etc. For temperature controla transparent stage may be used to heat the chip to a desiredtemperature.

Both the fluid comprising the droplets and the fluid carrying thedroplets (i.e., the aqueous and non-polar fluids) may have a relativelylow Reynolds Number, for example 10-2. The Reynolds Number represents aninverse relationship between the density and velocity of a fluid and itsviscosity in a channel of given length. More viscous, less dense, slowermoving fluids over a shorter distance will have a lower Reynolds Number,and are easier to divert, stop, start, or reverse without turbulence.Because of the small sizes and slow velocities, microfabricated fluidsystems are often in a low Reynolds number regime (Re<<1). In thisregime, inertial effects, which cause turbulence and secondary flows,are negligible; viscous effects dominate the dynamics. These conditionsare advantageous for sorting, and are provided by microfabricateddevices of the disclosure. Accordingly, the microfabricated devices ofthe disclosure are optionally operated at a low or very low Reynold'snumber.

In one aspect provided herein is a microfluidic device designed fordroplet based encoded library screening. In some embodiments, the devicecomprises a first microfluidic channel comprising an aqueous fluid. Insome embodiments, the device comprises a second microfluidic channelcomprising a fluid immiscible with the aqueous stream. In someembodiments, the device comprises a junction at which the firstmicrofluidic channel is in fluid communication with the secondmicrofluidic channel. In some embodiments, the junction of the first andsecond microfluidic channels defines a device plane. In someembodiments, the junction is configured to form droplets of the aqueousfluid within the fluid from the second microfluidic channel. In someembodiments, the second microfluidic channel is configured to continuepast the junction thereby defining an assay flow path. In someembodiments, the fluid from the second microfluidic channel with thedroplets therein moves past the junction in a third microfluidic channelthat defines an assay flow path. The assay flow path may also be calledan incubation region. In some embodiments, the device comprises acleavage region for cleaving effectors from scaffolds disposed withinthe assay flow path. In some embodiments, the device comprises adetection region. In some embodiments, the device comprises a sortingregion. In some embodiments, the device comprises a stimulation region.

In some embodiments, the device comprises a third microfluidic channel.The third microfluidic channel may be in fluidic communication with thefirst microfluidic channel upstream of the junction of the first andsecond microfluidic channels. This third microfluidic channel may beused to mix an additional aqueous fluid with the first aqueous fluidprior to droplet formation, thus allowing the mixing of different setsof reagents shortly before the droplets are formed.

The junction of the first and second microfluidic channels is configuredto create aqueous droplets encapsulated in the immiscible fluid of thesecond microfluidic channel. This junction may be of any configuration.In some embodiments, the junction is a T-junction. In some embodiments,the junction is at an oblique angle. In some embodiments, the junctionfurther comprises a supplementary microfluidic channel. In someembodiments, the supplementary microfluidic channel comprises a secondfluid immiscible with the aqueous stream. In some embodiments, thesecond fluid immiscible with the aqueous stream is the same as the fluidimmiscible with the aqueous stream from the second microfluidic channel.In some embodiments, the second fluid immiscible with the aqueous streamis different from the fluid immiscible with the aqueous stream from thesecond microfluidic channel. In some embodiments, the secondmicrofluidic channel and the supplementary microfluidic channel arepositioned on opposite sides of the first microfluidic channel. In someembodiments, the second microfluidic channel and the supplementarymicrofluidic channel are configured to add their respective fluidsimmiscible with the aqueous stream simultaneously.

After the junction, the flow path of the second microfluidic channel maycontinue along the same trajectory for a least a short distance. Afterdroplet formation, the channel downstream of the junction forms an assayflow path. The assay flow path is the path of the microfluidic channelwhere the screening assay is performed in the droplet. As the dropletcontinues along this assay flow path, additional unit operations can beperformed on the droplet in sequences that allow an assay with adetectable readout to occur within the droplet. In some embodiments, theassay flow path comprises a cleavage region. In some embodiments, theassay flow path comprises a detection region. In some embodiments, theassay flow path comprises a sorting region. In some embodiments, theassay flow path comprises a stimulation region.

The assay flow path may be in any shape. In some embodiments, the assayflow path acts as an incubation region, allowing the assay to beperformed over a desired length of time. In some embodiments, the assayflow path comprises a serpentine path region. The serpentine path regionmay contain a plurality of curves or turns. Such a pathway allows for anextended flow path to able to be embedded on a device of a small size.Additionally, the curves of the flow path may be used to orient variousdetectors, stimulators, sorters, or other components in a manner thatminimizes background signal, cross-talk, or bleed through of variousinputs into the droplets as they travel along the path. In someembodiments, this is accomplished by orienting the various inputs ofunit operations along the curves or turns of the serpentine path. Thisminimizes the amount of the input that can travel along the flow path.For example, configuring a light source to input the light at a locationalong a curve or turn of the flow path minimizes the light that willtravel along the path and reach droplets not the target of the emission.

The serpentine path region can be any length of the microfluidic deviceand can comprise any number of curves or turns. In some embodiments, theserpentine flow path region comprises about 10 curves to about 100curves. In some embodiments, the serpentine flow path region comprisesabout 10 curves to about 20 curves, about 10 curves to about 30 curves,about 10 curves to about 40 curves, about 10 curves to about 50 curves,about 10 curves to about 100 curves, about 20 curves to about 30 curves,about 20 curves to about 40 curves, about 20 curves to about 50 curves,about 20 curves to about 100 curves, about 30 curves to about 40 curves,about 30 curves to about 50 curves, about 30 curves to about 100 curves,about 40 curves to about 50 curves, about 40 curves to about 100 curves,or about 50 curves to about 100 curves. In some embodiments, theserpentine flow path region comprises about 10 curves, about 20 curves,about 30 curves, about 40 curves, about 50 curves, or about 100 curves.In some embodiments, the serpentine flow path region comprises at leastabout 10 curves, about 20 curves, about 30 curves, about 40 curves, orabout 50 curves. In some embodiments, the serpentine flow path regioncomprises at most about 20 curves, about 30 curves, about 40 curves,about 50 curves, or about 100 curves.

In some embodiments, the assay flow path comprises one or more chambersdisposed within the assay flow path. In some embodiments, one or more ofthe chambers comprise an entrance and exit microfluidic channel. In someembodiments, the entrance microfluidic channel is at an upstreamposition and the exit microfluidic channel is at a downstream positionof the chamber. In some embodiments, the entrance microfluidic channelsand exit microfluidic channels act as connecting channels betweenchambers. In some embodiments, each droplet travelling through the assayflow path travels through the one or more chambers. In some embodiments,the chambers are configured to adjust the flow rate of the droplets asthey flow through the assay flow path. In some embodiments, the chambersare configured to adjust the residence time of the droplets as they flowthrough the assay flow path. In some embodiments, the one or morechambers do not comprise an entrance and exit microfluidic channel(e.g., there are no connecting channels between the chambers). In someembodiments, the one or more chambers are connected to each other. Insome embodiments, the one or more chambers are arranged to formserpentine assay flow path.

Additional design considerations may be taken into mind when selectingdesired chamber and assay flow path geometry. For example,characteristics of the immiscible carrier fluid can influencesuitability of a chamber or channel geometry for a particular assaybeing performed on a device. For example, immiscible carrier fluids withhigh viscosity contribute to greater resistance to flow on the device,and thus are less compatible with device flow path geometries whichutilize substantial lengths of narrow channels or chambers. However,widening of channels or chambers on the device can increase dispersionof droplets travelling through the chambers or channels, therebyyielding a high variance of incubation times for individual dropletstravelling through the device. Thus, in some embodiments, it ispreferable that a device be operated with a low-viscosity immisciblecarrier fluid, such as 3-ethoxyperfluoro(2-methylhexane). In someembodiments, the device is designed to optimize characteristics such asresidence time, modest flow pressures, and dispersion ratio with aparticular immiscible carrier fluid. In some embodiments, the device isdesigned for optimal performance with low-density (e.g. less than 1.00g/mL) immiscible carrier fluid with low viscosity. In some embodiments,the device is designed for optimal performance with3-ethoxyperfluoro(2-methylhexane) as the immiscible carrier fluid.

In some embodiments, the chambers are configured to prevent the trappingof droplets as the droplets travel through the flow path. In embodimentswherein a carrier fluid denser than the aqueous droplets is used (e.g.3-ethoxyperfluoro(2-methylhexane)), aqueous droplets may rise to the topof the widened, heightened chambers and become trapped within thechamber as the droplets and carrier fluid flow through the device. Tocounteract this, in some embodiments, the chambers and entrance and orexit microfluidic channels are configured to have only a smalldifference in channel height between the chambers and the connectingchannels. In some embodiments, the height between the chambers and theexit channels does not change until after the width of the channel hasbeen narrowed along the flow path. By adjusting the height only afternarrowing the width of the channel, droplets are more prone to flowingalong the desired path and not becoming trapped.

In some embodiments, the height of the chambers is only slightly greaterthan the height of the connecting channels. In some embodiments, theheight of the chamber is about 1.1× to about 3× greater than the heightof the connecting channel. In some embodiments, the height of thechamber is about 3× to about 2.5×, about 3× to about 2×, about 3× toabout 1.9×, about 3× to about 1.8×, about 3× to about 1.7×, about 3× toabout 1.6×, about 3× to about 1.5×, about 3× to about 1.4×, about 3× toabout 1.3×, about 3× to about 1.2×, about 3× to about 1.1×, about 2.5×to about 2×, about 2.5× to about 1.9×, about 2.5× to about 1.8×, about2.5× to about 1.7×, about 2.5× to about 1.6×, about 2.5× to about 1.5×,about 2.5× to about 1.4×, about 2.5× to about 1.3×, about 2.5× to about1.2×, about 2.5× to about 1.1×, about 2× to about 1.9×, about 2× toabout 1.8×, about 2× to about 1.7×, about 2× to about 1.6×, about 2× toabout 1.5×, about 2× to about 1.4×, about 2× to about 1.3×, about 2× toabout 1.2×, about 2× to about 1.1×, about 1.9× to about 1.8×, about 1.9×to about 1.7×, about 1.9× to about 1.6×, about 1.9× to about 1.5×, about1.9× to about 1.4×, about 1.9× to about 1.3×, about 1.9× to about 1.2×,about 1.9× to about 1.1×, about 1.8× to about 1.7×, about 1.8× to about1.6×, about 1.8× to about 1.5×, about 1.8× to about 1.4×, about 1.8× toabout 1.3×, about 1.8× to about 1.2×, about 1.8× to about 1.1×, about1.7× to about 1.6×, about 1.7× to about 1.5×, about 1.7× to about 1.4×,about 1.7× to about 1.3×, about 1.7× to about 1.2×, about 1.7× to about1.1×, about 1.6× to about 1.5×, about 1.6× to about 1.4×, about 1.6× toabout 1.3×, about 1.6× to about 1.2×, about 1.6× to about 1.1×, about1.5× to about 1.4×, about 1.5× to about 1.3×, about 1.5× to about 1.2×,about 1.5× to about 1.1×, about 1.4× to about 1.3×, about 1.4× to about1.2×, about 1.4× to about 1.1×, about 1.3× to about 1.2×, about 1.3× toabout 1.1×, or about 1.2× to about 1.1× greater than the height of theconnecting channel. In some embodiments, the height of the chamber isabout 3×, about 2.5×, about 2×, about 1.9×, about 1.8×, about 1.7×,about 1.6×, about 1.5×, about 1.4×, about 1.3×, about 1.2×, or about1.1×. In some embodiments, the height of the chamber is at least about3×, about 2.5×, about 2×, about 1.9×, about 1.8×, about 1.7×, about1.6×, about 1.5×, about 1.4×, about 1.3×, or about 1.2× greater than theheight of the connecting channel. In some embodiments, the height of thechamber is at most about 2.5×, about 2×, about 1.9×, about 1.8×, about1.7×, about 1.6×, about 1.5×, about 1.4×, about 1.3×, about 1.2×, orabout 1.1× greater than the height of the connecting channel. In someembodiments, the height of the chamber is from about 1.1× to about 1.8×greater than the height of the connecting channel. In some embodiments,the height of the chamber is from about 1.4× to about 1.8× greater thanthe height of the connecting channel. In some embodiments, the height ofthe chamber is about 1.1× greater than the height of the connectingchannel. In some embodiments, the height of the chamber is about 1.2×greater than the height of the connecting channel. In some embodiments,the height of the chamber is about 1.3× greater than the height of theconnecting channel. In some embodiments, the height of the chamber isabout 1.4× greater than the height of the connecting channel. In someembodiments, the height of the chamber is about 1.5× greater than theheight of the connecting channel. In some embodiments, the height of thechamber is about 1.6× greater than the height of the connecting channel.In some embodiments, the height of the chamber is about 1.7× greaterthan the height of the connecting channel. In some embodiments, theheight of the chamber is about 1.8× greater than the height of theconnecting channel. In some embodiments, the height of the chamber isabout 1.9× greater than the height of the connecting channel. In someembodiments, the height of the chamber is about 2× greater than theheight of the connecting channel.

In some embodiments, the height of the chamber is about 50 microns toabout 120 microns. In some embodiments, the height of the chamber isabout 120 microns to about 100 microns, about 120 microns to about 90microns, about 120 microns to about 80 microns, about 120 microns toabout 70 microns, about 120 microns to about 60 microns, about 120microns to about 50 microns, about 100 microns to about 90 microns,about 100 microns to about 80 microns, about 100 microns to about 70microns, about 100 microns to about 60 microns, about 100 microns toabout 50 microns, about 90 microns to about 80 microns, about 90 micronsto about 70 microns, about 90 microns to about 60 microns, about 90microns to about 50 microns, about 80 microns to about 70 microns, about80 microns to about 60 microns, about 80 microns to about 50 microns,about 70 microns to about 60 microns, about 70 microns to about 50microns, or about 60 microns to about 50 microns. In some embodiments,the height of the chamber is about 120 microns, about 100 microns, about90 microns, about 80 microns, about 70 microns, about 60 microns, orabout 50 microns. In some embodiments, the height of the chamber is atleast about 120 microns, about 100 microns, about 90 microns, about 80microns, about 70 microns, or about 60 microns. In some embodiments, theheight of the chamber is at most about 100 microns, about 90 microns,about 80 microns, about 70 microns, about 60 microns, or about 50microns. In some embodiments, the height of the chamber is about 80microns.

In some embodiments, the height of the chamber is about 300 microns toabout 1,000 microns. In some embodiments, the height of the chamber isabout 1,000 microns to about 750 microns, about 1,000 microns to about600 microns, about 1,000 microns to about 500 microns, about 1,000microns to about 450 microns, about 1,000 microns to about 400 microns,about 1,000 microns to about 350 microns, about 1,000 microns to about300 microns, about 750 microns to about 600 microns, about 750 micronsto about 500 microns, about 750 microns to about 450 microns, about 750microns to about 400 microns, about 750 microns to about 350 microns,about 750 microns to about 300 microns, about 600 microns to about 500microns, about 600 microns to about 450 microns, about 600 microns toabout 400 microns, about 600 microns to about 350 microns, about 600microns to about 300 microns, about 500 microns to about 450 microns,about 500 microns to about 400 microns, about 500 microns to about 350microns, about 500 microns to about 300 microns, about 450 microns toabout 400 microns, about 450 microns to about 350 microns, about 450microns to about 300 microns, about 400 microns to about 350 microns,about 400 microns to about 300 microns, or about 350 microns to about300 microns. In some embodiments, the height of the chamber is about1,000 microns, about 750 microns, about 600 microns, about 500 microns,about 450 microns, about 400 microns, about 350 microns, or about 300microns. In some embodiments, the height of the chamber is at leastabout 1,000 microns, about 750 microns, about 600 microns, about 500microns, about 450 microns, about 400 microns, or about 350 microns. Insome embodiments, the height of the chamber is at most about 750microns, about 600 microns, about 500 microns, about 450 microns, about400 microns, about 350 microns, or about 300 microns. In someembodiments, the height of the chamber is about 500 microns.

In some embodiments, the width of the connecting channel is about 50microns to about 120 microns. In some embodiments, the width of theconnecting channel is about 120 microns to about 100 microns, about 120microns to about 90 microns, about 120 microns to about 80 microns,about 120 microns to about 70 microns, about 120 microns to about 60microns, about 120 microns to about 50 microns, about 100 microns toabout 90 microns, about 100 microns to about 80 microns, about 100microns to about 70 microns, about 100 microns to about 60 microns,about 100 microns to about 50 microns, about 90 microns to about 80microns, about 90 microns to about 70 microns, about 90 microns to about60 microns, about 90 microns to about 50 microns, about 80 microns toabout 70 microns, about 80 microns to about 60 microns, about 80 micronsto about 50 microns, about 70 microns to about 60 microns, about 70microns to about 50 microns, or about 60 microns to about 50 microns. Insome embodiments, the width of the connecting channel is about 120microns, about 100 microns, about 90 microns, about 80 microns, about 70microns, about 60 microns, or about 50 microns. In some embodiments, thewidth of the connecting channel is at least about 120 microns, about 100microns, about 90 microns, about 80 microns, about 70 microns, or about60 microns. In some embodiments, the width of the connecting channel isat most about 100 microns, about 90 microns, about 80 microns, about 70microns, about 60 microns, or about 50 microns. In some embodiments, thewidth of the connecting channel is about 80 microns.

In some embodiments, the height of the connecting channel is about 35microns to about 75 microns. In some embodiments, the height of theconnecting channel is about 75 microns to about 65 microns, about 75microns to about 55 microns, about 75 microns to about 50 microns, about75 microns to about 45 microns, about 75 microns to about 40 microns,about 75 microns to about 35 microns, about 65 microns to about 55microns, about 65 microns to about 50 microns, about 65 microns to about45 microns, about 65 microns to about 40 microns, about 65 microns toabout 35 microns, about 55 microns to about 50 microns, about 55 micronsto about 45 microns, about 55 microns to about 40 microns, about 55microns to about 35 microns, about 50 microns to about 45 microns, about50 microns to about 40 microns, about 50 microns to about 35 microns,about 45 microns to about 40 microns, about 45 microns to about 35microns, or about 40 microns to about 35 microns. In some embodiments,the height of the connecting channel is about 75 microns, about 65microns, about 55 microns, about 50 microns, about 45 microns, about 40microns, or about 35 microns. In some embodiments, the height of theconnecting channel is at least about 75 microns, about 65 microns, about55 microns, about 50 microns, about 45 microns, or about 40 microns. Insome embodiments, the height of the connecting channel is at most about65 microns, about 55 microns, about 50 microns, about 45 microns, about40 microns, or about 35 microns. In some embodiments, the height of theconnecting channel is about 50 microns.

In some embodiments, the chambers are configured to reduce the flow rateof the droplets as the droplets travel through the device. In someembodiments, the flow rate is reduced due to an increase in thecross-sectional area of the chamber relative to the microfluidic channelupstream of the chamber. For example, a chamber having 10× thecross-sectional area compared to the microfluidic channel upstream ofthe chamber would have a flow rate through the chamber of 10% of theflow rate compared to the flow rate through the upstream microfluidicchannel. In some embodiments, the flow rate through the chambers isabout 1% to about 25% of the flow rate through the microfluidic channelupstream of the chambers. In some embodiments, the flow rate through thechambers is about 25% to about 20%, about 25% to about 15%, about 25% toabout 12%, about 25% to about 10%, about 25% to about 8%, about 25% toabout 5%, about 25% to about 3%, about 25% to about 1%, about 20% toabout 15%, about 20% to about 12%, about 20% to about 10%, about 20% toabout 8%, about 20% to about 5%, about 20% to about 3%, about 20% toabout 1%, about 15% to about 12%, about 15% to about 10%, about 15% toabout 8%, about 15% to about 5%, about 15% to about 3%, about 15% toabout 1%, about 12% to about 10%, about 12% to about 8%, about 12% toabout 5%, about 12% to about 3%, about 12% to about 1%, about 10% toabout 8%, about 10% to about 5%, about 10% to about 3%, about 10% toabout 1%, about 8% to about 5%, about 8% to about 3%, about 8% to about1%, about 5% to about 3%, about 5% to about 1%, or about 3% to about 1%of the flow rate through the microfluidic channel upstream of thechambers. In some embodiments, the flow rate through the chambers isabout 25%, about 20%, about 15%, about 12%, about 10%, about 8%, about5%, about 3%, or about 1% of the flow rate through the microfluidicchannel upstream of the chambers. In some embodiments, the flow ratethrough the chambers is at least about 25%, about 20%, about 15%, about12%, about 10%, about 8%, about 5%, or about 3%. In some embodiments,the flow rate through the chambers is at most about 20%, about 15%,about 12%, about 10%, about 8%, about 5%, about 3%, or about 1% of theflow rate through the microfluidic channel upstream of the chambers. Insome embodiments, the flow rate through the chambers is about 10% of theflow rate through the microfluidic channel upstream of the chambers. Insome embodiments, the flow rate through the microfluidic channelupstream of the chambers varies at different points of the device. Insuch embodiments, the flow rate used for the flow rate comparison to thechambers is the fastest flow rate after the droplet formation junction(e.g. the microfluidic channel portion with the smallest cross-sectionalarea).

In some embodiments, the chambers are configured such that the dropletsformed on the microfluidic device have substantially the same residencetime travelling through the device. In some embodiments, themicrofluidic device is configured such that the droplets form on thedevice have substantially the same residence time travelling through thedevice. In some embodiments, this is measured by the dispersion ratio.The dispersion ratio is calculated according to the following formula:6σ/T_(avg); wherein T_(avg) is the average residence time of a droplettravelling through the device and σ is the standard deviation ofresidence time of droplets travelling through the device. In someembodiments, the device has a dispersion ratio of at most about 10%,about 8%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1%.In some embodiments, the device has a dispersion ratio of at most about10%. In some embodiments, the device has a dispersion ratio of at mostabout 8%. In some embodiments, the device has a dispersion ratio of atmost about 6%. In some embodiments, the device has a dispersion ratio ofat most about 5%. In some embodiments, the device has a dispersion ratioof at most about 4%. In some embodiments, the device has a dispersionratio of at most about 3%. In some embodiments, the device has adispersion ratio of at most about 2%. In some embodiments, the devicehas a dispersion ratio of at most about 1%.

FIG. 18. provides an exemplary data set depicting a level of uniformityfor incubation period using a microfluidic device as depicted FIGS. 9Aand 10. Specifically, two aqueous inputs were provided to inlets 101,102, wherein one aqueous input contained a buffer solution andfluorophore (“fluorophore solution”), and the other aqueous inputcontained just the buffer solution (“buffer solution”). The fluorophoresolution and buffer solution were provided with setpoint pressures thatwere offset by about 3%, such that one solution would flow through theassay flow channel with a higher concentration than the other.Initially, the buffer solution was provided with the higher pressure,after which the setpoint pressures were switched such that thefluorophore solution was provided at a higher pressure. As depicted inFIG. 18, the PMT count for a first period of time is less than 100 rfuafter which there is a sudden increase. The dispersion amongst the dataset was calculated to be only 1.7%, with a sigma of 3.19. As such, thisdisplays a level of uniform incubation period as the fluorophoresolution provided detection signals within 2% dispersion, and withoutsignificant lag when switching the concentrations. As such, thiscorrelates to encapsulations moving along the assay flow path at arelatively uniform rate. FIG. 19 provides a similar analysis using themicrofluidic device from FIG. 11, wherein the fluorophore solution wasprovided with a higher pressure initially, before being switched to alower pressure. The dispersion amongst the data point was calculated tobe slightly higher at 4.52%, with a sigma of 7.25. As such, thissimilarly displays a level of uniformity for the incubation period asthe fluorophore solution provided detection signals with less than 5%dispersion.

In some embodiments, the device further comprises one or more collectionchambers. In some embodiments, the one or more collection chambers areconfigured to receive a subset of the plurality of droplets passingthrough the assay flow path. In some embodiments, the collectionchambers are configured to incubate the subset for an extended period oftime. In some embodiments, the collection chambers are configured tolengthen the residence time for the subset of plurality of droplets.

In some embodiments, the device further comprises one or more shuntspositioned along the flow path of the device. A shunt may be positionedat any location of the device. The shunt may be used for a variety ofpurposes. In some embodiments, a shunt is used to insert additionalimmiscible carrier fluid into the microfluidic channel in order toaffect droplet spacing. In some embodiments, a shunt is used to divertdroplets of carrier fluid off of the microfluidic device. In someembodiments, a shunt is used in initiation of the device. In someembodiments, a shunt is used in equilibration of the device. In someembodiments, the device is equilibrated

In some embodiments, the assay flow path comprises a first shunt. Insome embodiments, the first shunt is positioned in an upstream area ofthe assay flow path. In some embodiments, the first shunt is positionedupstream of the serpentine area of the assay flow path. In someembodiments, the first shunt is positioned upstream of the one or morechambers. In some embodiments, the first shunt is opened during anequilibration phase of using the device. In some embodiments, carrierfluid is run through the device in a reverse direction from normaloperation during an equilibration stage of the device and allowed toexit the device through the first shunt. In some embodiments, aqueousdroplets are simultaneously introduced into the microfluidic deviceupstream of the first shunt and allowed to exit the device through thefirst shunt. In some embodiments, the shunt is closed once pressures ofinput fluids on the device have been adjusted to desired levels in orderto run the system as desired (e.g. flow rates, pressures, droplet size,droplet spacing, etc.).

In some embodiments, the first shunt configured to allow droplets tobypass at least a portion of the assay flow path. In some embodiments,an alternate flow path is coupled to the first shunt. The alternate flowpath can have any property and can be used to affect the assay flow pathin any manner. For example, the alternate flow path can be used tochange the incubation time or residence time of droplets on themicrofluidic device, add an additional reagent steam (e.g. a dropletmerging junction or pico-injection site), or to incubate droplets offthe device entirely.

The cleavage region may comprise a mechanism for liberating an effectorthat is linked to a bead by a cleavable linker. In some embodiments, thecleavage region comprises a pico-injection site or droplet merging siteto introduce reagents to cleave the effector from a scaffold. In someembodiments, the cleavage region comprises a light source configured tocleave effectors from scaffolds disposed within the assay flow path. Insome embodiments, the light source is a source of UV light. In someembodiments, the light source is a waveguide. In some embodiments, thelight source is a fiberoptic cable. In some embodiments, the lightsource is a light source configured to cleave effectors from scaffoldsdisposed within the assay flow path. In some embodiments, the lightsource is configured to have an optical axis substantially parallel withthe device plane. In some embodiments, the light source illuminates apassing droplet at a curve in the assay flow path. In some embodiments,the light source is configured to have an optical axis substantiallyperpendicular to the device plane. In some embodiments, the light sourceis aligned with the microfluidic channel of the cleavage region bypillars mounted on the device. In some embodiments, the light source isconfigured to emit light over an area covering multiple portions of themicrofluidic channel passing through the cleavage region. In someembodiments, the cleavage region comprises a serpentine flow path.

The cleavage region can be at any point along the microfluidic devicedepending upon the needs of the assay being employed on the device. Insome embodiments, the cleavage region is upstream of the detectionregion, the sorting region, and the stimulation region. In someembodiments, the cleavage region is upstream of the detection region. Insome embodiments, the cleavage region is upstream of the sorting region.In some embodiments, the cleavage region is upstream of the stimulationregion. In some embodiments, the cleavage region is upstream of thedetection region and the sorting region.

In some embodiments, the device comprises an additional inlet and outletpositioned on the microfluidic channel upstream and downstream of thecleavage region. In some embodiments, the inlet and outlets arepositioned immediately before and immediately after the cleavage region.

In some embodiments, these inlets and outlets are configured to allowfor a calibration of the cleavage region. The calibration allows forcontrol over device-to-device variability in how much light the samplespassing through the cleavage region are exposed to. Such variability cancome from small changes to a variety of parameters of the device,including the coupling of the light source to the device. Variability inexposure intensity time and duration can lead to variability in amountof compound released from beads, which can cause errors in ultimatescreening assay readouts.

In some embodiments, the inlets and outlets are used for the calibrationprocedure. In some embodiments, the calibration procedure comprisesflowing a solution comprising a fluorescent dye through the cleavageregion. FIG. 12D provides an exemplary depiction of the cleavage regionfor a microfluidic device described herein, wherein the calibrationinlet and UV waveguide for exposing the encapsulations (e.g., droplets)to light are shown. In some embodiments, the calibrant channel is filledwith UV-sensitive fluorophore to measure the UV intensity in thecleavage region. In some embodiments, the UV waveguide directs lightfrom a UV LED coupled fiberoptic into a confined area. In someembodiments, the UV LED power is then set, based on a calibrant dyebeing measured. FIG. 12E provides exemplary data correlating a calibrantdye with a given light exposure (BD Horizon™ BV510).

In some embodiments, encoded effector-fluorophore beads are introducedinto encapsulations (e.g., droplets) using a microfluidic device asdescribed herein. FIG. 12A provides an exemplary solution of beads withan encoded-effector modified with a fluorophore, wherein the solutioncan comprise a library of beads. As shown in FIG. 12B, theeffector-fluorophore may be connected by a photo-cleavable, orpro-photo-cleavable linker. In some embodiments, the encoded-fluorophorebeads are introduced into droplets at approximately 200 pL in volume. Insome embodiments, the droplets are introduced into the cleavage regionand exposed to the UV light. As shown in FIG. 12B, when exposed to theUV light, the effector is liberated (i.e. cleaving the photocleavablelinker), such that the effector is released from the bead (FIG. 12C).The droplets then continue to flow through the microfluidic device, asdescribed herein, until reaching an “interrogation region” of themicrofluidic device, wherein the droplets are subject to laserexcitation (e.g., confocal laser excitation, FIG. 12F), thereby excitingthe released effector-fluorophore. The emission from the encodedeffector-fluorophore is then collected by PMT detectors, as shown inFIGS. 13A and 14A (represented by PMT 2 Smooth), which represent theeffector release based on exposure from 100 mV and 600 mV lightrespectively, thereby measuring the released effector-fluorophoreconcentration. FIGS. 13B and 14B provide the peak emission measured foreach droplet based on exposure from 100 mV and 600 mV lightrespectively, plotted as a heat-map over time to observe the stabilityof the signal. As shown, increasing the UV LED power increases theexposure, thereby enabling the ability to control the finalconcentration of released effector-fluorophore. FIG. 15B provides ahistogram with compressed droplet maps (e.g., from FIGS. 13B and 14B),so as to depict normally distributed intensity values. The median valueis correlated to known Fluorophore concentration calibrations (e.g.,FIG. 15A), so as to determine the final concentration of theeffector-fluorophore after UV release. As such, the emission intensity,as measured with a calibrant fluid, can be correlated to a resultanteffector-release concentration, thereby providing a predictivequantitative release.

The detection region is configured with a detector capable of detectingany desired readout of an assay to be performed on the device. In someembodiments, the detection region comprises a fluorometer. In someembodiments, the fluorometer comprises a photomultiplier tube detector,a light source, an excitation filter and an emission filter. In someembodiments, the fluorometer is configured to have an optical axissubstantially parallel to the device plane. In some embodiments, thefluorometer is configured to have an optical axis substantiallyperpendicular to the device plane. In some embodiments, the fluorometerilluminates a passing droplet at a curve in the assay flow path. In someembodiments, the detection region comprises confocal detection and laserscanning. In some embodiments, the detection region comprises a confocallaser scanning device, as shown in FIGS. 22A-B (providing a top view ofthe device). FIG. 12F provides an exemplary schematic of encapsulationdetection via confocal laser scanning. In some embodiments, thedetection region comprises laser scanning. In some embodiments, thedetection region comprises fluorescence. In some embodiments, thedetection region comprises any combination of detection means describedherein.

In some embodiments, the detection region comprises an objective orfiber for emitting an excitation light into the detection region. Insome embodiments, the detection region comprises an objective, fiber, orcharged coupled device configured to collect emission from the detectionregion. In some embodiments, a single objective is configured to directexcitation and collect emission from the detection region. In someembodiments, the objective configured to collect emission from thedetection region (which may be the same as the excitation objective) isan inverted objective lens. In some embodiments, the objectiveconfigured to collect emission from the detection region (which may bethe same as the excitation objective) is configured to collect,collimate, and direct the emitted light through optical fibers. In someembodiments, the optical fibers are coupled to a detector configured toquantify the emission. In some embodiments, the detector configured toquantify the emission is a photomultiplier tube, charged coupled device,or photodiode.

In some embodiments, the detection region is capable of being moved onthe chip. In some embodiments, the detection region comprises anexcitation light source that is not coupled to the device. In someembodiments, the detection region comprises an objective that is notcoupled to the device. In some embodiments, having a light source ordetector for the detection region not coupled to the device allows forthe system to be adjusted based on assay need. For example, the systemcan be adjusted to increase or decrease the time between detection andsorting. Additionally, the system can be adjusted so that a single lightsource may be used for calibration and initialization of the deviceprior to performing a screening assay on the device.

In some embodiments, the detection region is configured to detect two ormore wavelengths of fluorescence. This allows for the detection of theabundance of a plurality of fluorescent probes. In some embodiments, thedroplet being assayed may comprise a control fluorophore and an assayfluorophore. The assay fluorophore gives a readout of the assay, e.g. apositive or negative result of the assay. The control fluorophore, ifpresent, may be detected and quantified. In some embodiments, thecontrol fluorophore is placed into the aqueous fluid of the firstmicrofluidic channel at a known concentration. When the dropletcomprising the aqueous fluid of the first microfluidic channel reachesthe detection region, the amount of control fluorophore fluorescencedetected can be used to quantify the size of the droplet. This can beused to normalize the results of the assay fluorophore readout. In someembodiments, the detection region is configured to measure two or moreassay fluorophores.

In some embodiments, the device comprises a single detection region. Insome embodiments, the detection region is downstream of the cleavageregion. In some embodiments, the detection region is downstream of thestimulation region. In some embodiments, the detection region isupstream of the sorting region.

In some embodiments, the device comprises multiple detection regions.When the device comprises multiple detection regions, they may be placedanywhere on the device. In some embodiments, the detection region isconfigured to be in communication with another region. For example, thedetection region may be in communication with the sorting region toallow sorting to occur based on the detection of a signal. In someembodiments, a detection region is configured to be in communicationwith a pico-injector. When a detection region is in communication with apico-injector, reagents or other assay components can be selectivelyadded only when certain conditions are met, such as the presence orabsence of a signal.

In some embodiments, the device comprises a stimulation region. In someembodiments, the stimulation region comprises one or more actuators forstimulating an ion channel. Any method of stimulating an ion channel maybe employed by the actuators when the device is configured to perform anion channel modulation assay. In some embodiments, the stimulationregion comprises one or more actuators for stimulating an ion channel.In some embodiments, the one or more actuators for stimulating the ionchannel comprises at least one light source, at least one electrode, orat least one pico-injection site equipped with an ion channel toxin. Insome embodiments, the one or more actuators comprises at least one lightsource. In some embodiments, the one or more actuators comprises atleast one electrode. In some embodiments, the one or more actuatorscomprises an injection site for an ion channel toxin.

In some embodiments, the one or more actuators comprises at least oneelectrode. Any type of electrode capable of delivering anelectromagnetic current to the encapsulation may be employed. In someembodiments, the electrode lies along a wall of the assay flow path anddelivers an electric field to the passing stream. In some embodiments,the electric field is pulsed to match the frequency at which dropletspass the electrode.

In some embodiments, the one or more actuators comprises a pair ofelectrodes on opposite walls of the assay flow path such that when adroplet passes the pair of electrodes the droplet contacts theelectrodes, thereby allowing a current to flow through the droplet. Insome embodiments, the device comprises multiple pairs of electrodes soconfigured. In some embodiments, the stimulation region comprises about1 pair to about 20 pairs of electrodes so configured. In someembodiments, the stimulation region comprises about 1 pair to about 2pairs, about 1 pair to about 3 pairs, about 1 pair to about 5 pairs,about 1 pair to about 7 pairs, about 1 pair to about 10 pairs, about 1pair to about 20 pairs, about 2 pairs to about 3 pairs, about 2 pairs toabout 5 pairs, about 2 pairs to about 7 pairs, about 2 pairs to about 10pairs, about 2 pairs to about 20 pairs, about 3 pairs to about 5 pairs,about 3 pairs to about 7 pairs, about 3 pairs to about 10 pairs, about 3pairs to about 20 pairs, about 5 pairs to about 7 pairs, about 5 pairsto about 10 pairs, about 5 pairs to about 20 pairs, about 7 pairs toabout 10 pairs, about 7 pairs to about 20 pairs, or about 10 pairs toabout 20 pairs of electrodes so configured. In some embodiments, thestimulation region comprises about 1 pair, about 2 pairs, about 3 pairs,about 5 pairs, about 7 pairs, about 10 pairs, or about 20 pairs ofelectrodes so configured. In some embodiments, the stimulation regioncomprises at least about 1 pair, about 2 pairs, about 3 pairs, about 5pairs, about 7 pairs, or about 10 pairs of electrodes so configured. Insome embodiments, the stimulation region comprises at most about 2pairs, about 3 pairs, about 5 pairs, about 7 pairs, about 10 pairs, orabout 20 pairs of electrodes so configured.

Any number of actuators may be employed on the microfluidic device. Insome embodiments, the stimulation region comprises about 1 actuator toabout 20 actuators. In some embodiments, the stimulation regioncomprises about 1 actuator to about 2 actuators, about 1 actuator toabout 3 actuators, about 1 actuator to about 5 actuators, about 1actuator to about 7 actuators, about 1 actuator to about 10 actuators,about 1 actuator to about 20 actuators, about 2 actuators to about 3actuators, about 2 actuators to about 5 actuators, about 2 actuators toabout 7 actuators, about 2 actuators to about 10 actuators, about 2actuators to about 20 actuators, about 3 actuators to about 5 actuators,about 3 actuators to about 7 actuators, about 3 actuators to about 10actuators, about 3 actuators to about 20 actuators, about 5 actuators toabout 7 actuators, about 5 actuators to about 10 actuators, about 5actuators to about 20 actuators, about 7 actuators to about 10actuators, about 7 actuators to about 20 actuators, or about 10actuators to about 20 actuators. In some embodiments, the stimulationregion comprises about 1 actuator, about 2 actuators, about 3 actuators,about 5 actuators, about 7 actuators, about 10 actuators, or about 20actuators. In some embodiments, the stimulation region comprises atleast about 1 actuator, about 2 actuators, about 3 actuators, about 5actuators, about 7 actuators, or about 10 actuators. In someembodiments, the stimulation region comprises at most about 2 actuators,about 3 actuators, about 5 actuators, about 7 actuators, about 10actuators, or about 20 actuators.

In some embodiments, the device comprises multiple stimulation regions.Stimulation regions may be distributed in any orientation throughout themicrofluidic device. In some embodiments, the stimulation region isdownstream of the cleavage region. In some embodiments, the stimulationregion is upstream of the detection region. In some embodiments, thestimulation region is upstream of the sorting region.

In some embodiments, the device comprises an additional inlet configuredto insert carrier fluid into the flow path of the microfluidic device.Optimal spacing of droplets is an important consideration in order toaccurately sort desired droplets. Factors which can affect accuratesorting of droplets include droplet size, average separation ofdroplets, total oil fraction of the flow, ionic strength of thedroplets, and contents of the droplets. Individual assays performed onthe devices provided herein may require optimization of spacing, whichis allowed by the presence of the additional inlet. In some embodiments,the additional inlet inserts additional carrier fluid into the flow pathof the microfluidic device to increase spacing of the droplets. In someembodiments, the additional inlet inserts additional carrier fluid intothe flow path of the microfluidic device to focus the droplets. In someembodiments, the additional carrier fluid is the immiscible fluid fromthe second microfluidic channel. In some embodiments, the additionalcarrier fluid is different from the immiscible fluid from the secondmicrofluidic channel. In some embodiments, the additional inlet operatesat a constant flow. In some embodiments, the additional inlet operatesat a variable flow. In preferred embodiments, the additional inlet ispositioned shortly upstream of the detection region. In someembodiments, the additional inlet operates at a flow rate selected tooptimally space the droplets. In some embodiments, the device comprisestwo additional inlets. In some embodiments, the device comprises a firstadditional inlet configured to deliver spacing oil and a secondadditional inlet configured to deliver focusing oil.

In some embodiments, the devices comprise a sorting region. Any methodof sorting the droplets in the device may be used. In some embodiments,the sorting region is in communication with the detection region. Insome embodiments, the sorting region comprises a sorting apparatus thatsorts the droplets based on the detection of the presence, absence, orlevel of a signal detected by the detection region. In some embodiments,the sorting region comprises a sorting electrode. In some embodiments,the sorting electrode is an electrophoresis electrode. In someembodiments, the sorting electrode is a dielectrophoresis electrode. Insome embodiments, the sorting region comprises a valve configured forsorting. In some embodiments, the sorting region comprises a deflectablemembrane configured for sorting. In some embodiments, the sorting regioncomprises an acoustic wave generator configured for sorting. In someembodiments, the sorting region comprises an inlet for fluid configuredto guide a passing droplet down a sorted path.

In some embodiments, the device comprises microfluidic channels whichare fully enclosed. In some embodiments, the device comprisesmicrofluidic channels encompassed on all sides of the microfluidicchannel, except for any inlets and outlets into the device. In someembodiments, the device comprises a cover slip configured to enclose thechannels. In some embodiments, the cover slip is coated with ahydrophobic material (e.g. PDMS). The cover slip may be of any size(e.g. 5 micron, 10 micron, 15 micron, 20 micron, 30 micron, 40 micron,50 micron or greater).

Control of flow of fluids through the device may be accomplished in anymanner. In preferred embodiments, the flow of fluids is controlled by adevice capable of delivering fluid through the device for a prolongedperiod of time and/or in a continuous fashion (e.g. a pneumatic pump ora peristaltic pump). Such pumps have several advantages over otherpumps, such as syringe pumps, including the ability to run the systemfor a prolonged period of time at constant pressure, thus allowing forcontinuous feed of material through the device and control overresidence time of droplets travelling through the device. In someembodiments, the flow of fluids is controlled by a continuous pump. Insome embodiments, the flow of fluids is controlled by a pneumatic pump.In some embodiments, the fluids are delivered to the device from areservoir of fluid off of the device. This allows the device to draw amuch larger amount of fluid than would be possible from an on-devicereservoir. Any of the sample fluids, immiscible fluids, spacing oil,focusing oil, or other fluid delivered onto the chip can be delivered inthis manner.

In some embodiments, the pump is configured to deliver fluids throughthe device for a continuous period of at least 4 hours, at least 8hours, at least 12 hours, at least 16 hours, or at least 24 hours. Insome embodiments, the pump is configured to deliver fluids through thedevice for a continuous period of at least 12 hours. In someembodiments, the pump is configured to deliver fluids through the devicefor a continuous period of at least 24 hours.

A non-limiting, exemplary microfluidic device is shown in FIG. 9A. Theexemplary microfluidic device contains a first inlet 101. The firstinlet 101 is configured to accept an aqueous fluid, such as an aqueousassay reagent. The exemplary microfluidic device also contains a secondinlet 102. In this example, the second inlet 102 is configured to acceptanother aqueous fluid. This may be the same or different as the aqueousfluid added to the first inlet 101. The second inlet 102 may beconfigured to accept beads as provided herein, or the first inlet 101may be so configured. In other examples of a microfluidic device, theremay only be a single inlet stream. The exemplary microfluidic deviceshown in FIG. 9A further comprises an inlet 103 for carrier fluid (e.g.an oil immiscible with an aqueous fluid) in fluid connection with adroplet formation junction or extrusion junction 104. The inlet 103 inthis example is connected to the droplet formation junction 104 by twochannels, each reaching an aqueous stream channel at the same point onopposite sides of the aqueous stream channel. The droplet formationjunction 104 comprises a microfluidic channel that continues down theflow path towards cleavage region 106. Near cleavage region 106 is afiberoptic waveguide 105 a configured to deliver light into themicrofluidic channel of the cleavage region 106. The fiberopticwaveguide 5 a is embedded in the plane of the device such that the lightemitted enters the microfluidic channel of cleavage region 106 from thedevice plane. Also near cleavage region 106 is a pillar 105 b configuredto fix a fiberoptic manifold which can be configured to emit light fromabove the plane of the device into the microfluidic channel of cleavageregion 106. The light sources of 105 a and 105 b can be usedalternatively or in combination. The device also comprises an inlet forcalibration fluid 107 a in fluid connection with the cleavage region 106and an outlet for calibration fluid 107 b. The inlet for calibrationfluid 107 a is configured to receive and deliver to the cleavage region106 a fluid configured to normalize photon exposure within the cleavageregion. After passing through the cleavage region 106, the calibrationfluid exits through the outlet for calibration fluid 107 b. The cleavageregion 106 is in fluid communication via a microfluidic channel to anincubation region 109. In the example of FIG. 9A, the incubation region109 contains a series of widened chambers, each chamber connected to thenext chamber in the series by a microfluidic channel. The configurationof these chambers affect the flow rate and residence time of thedroplets formed at droplet formation region 104 through the device. Insome embodiments, the chambers are configured to prevent trapping ofdroplets as they pass through incubation region 109. Such configurationof the chambers is particularly important when using a carrier fluidthat is denser than the aqueous droplets (e.g.3-ethoxyperfluoro(2-methylhexane)). In some embodiments, this desiredconfiguration is achieved by configuring the chambers and connectingchannels to have only small difference in channel height between thechambers and the connecting channels. In some embodiments, the height ofthe chamber is about 80 μm and the height of the connecting channel isabout 50 μm. As an additional design feature to aid in prevention oftrapping of bubbles within the device, the height of the flow path doesnot change between the width of the chamber has been narrowed as thedroplet approaches the connecting channel, thus facilitating the smoothtransition of droplets from chamber to chamber without trapping.Configured on either end of incubation region 109 are bypass shunts 108a and 108 b. The bypass shunts 108 a and 108 b are configured to allow afluid coupled to the shunt to flow in or out of the main microfluidicchannel. If fluid is diverted out of the main microfluidic channel atbypass shunt 108 a, the material will not pass through incubation region109. Positioned downstream of incubation region 109 is inlet for carrierfluid 110. Inlet for carrier fluid 110 is in fluid communication withthe main microfluidic channel of the device and is configured to deliveradditional immiscible carrier fluid into the main microfluidic channelin order to space droplets as desired. Also in fluid communication withthe main microfluidic channel is inlet for carrier fluid 111, which isconfigured to deliver droplet focusing oil into the main microfluidicchannel. Downstream of inlets for carrier fluid 110 and 111 is detectionposition 116. The detection position 116 indicates the point on thedevice that the desired signal from the assay being run on the chip isdetected. The detection position 116 may be based on an alignment of anobjective or fiber that directs an excitation light at the samplepassing detection position 116 and an additional objective or fibercoupled to a detector configured to detect an emission from detectionposition 116. Alternatively, the objective for the excitation light maybe configured to also collect the emission. In some embodiments, theexcitation source is reflected from detection position 116 through aninverted objective lens, where the emission is collected, columnated,and directed through optical fibers for quantification by aphotomultiplier tube or other detector. In some embodiments, theobjective or fiber aligned at detection position 116 is not coupled tothe device. When not coupled to the device, the detector or emissionobjective or fiber can be moved to adjust the detection positions 116 onthe device in order to adjust the time between detection and sorting.When not coupled to the device, the detector or emission objective mayalso be moved for use in calibration of the device or initiation of thedevice, thus allowing a single light source to be used for multiplefunctions. Downstream of inlets for carrier fluid 110 and 111 anddetection position 116 is discrimination junction electrode 112. Thediscrimination junction electrode 112 may be a dielectrophoresiselectrode configured to propel droplets down outlet 114 if the dropletis determined to display a desired signal or to outlet 115 if thedroplet is determined to lack a desired signal. The discriminationjunction electrode 112 is connected to a discrimination junction groundcircuit, which is connected to the device at circuit connection points113 a and 113 b. A zoomed in drawing of the sorting and detection regionof the exemplary device is shown in FIG. 9B. FIG. 9C shows a picture ofa microfluidic device substantially as described in this example. FIG.10 provides another exemplary depiction of the microfluidic device fromFIG. 9A, wherein an Optical Glue is displayed within the fiberopticwaveguide. In some embodiments, the Optical Glue helps to minimizescattering of the light from the fiberoptic wave guide.

FIG. 11 provides another exemplary microfluidic device that can be usedfor the methods and systems described herein. The exemplary microfluidicdevice contains a first inlet 201. The first inlet 201 is configured toaccept an aqueous fluid, such as an aqueous assay reagent. The exemplarymicrofluidic device also contains a second inlet 202. In this example,the second inlet 202 is configured to accept another aqueous fluid. Thismay be the same or different as the aqueous fluid added to the firstinlet 201. The second inlet 202 may be configured to accept beads asprovided herein, or the first inlet 201 may be so configured. In someembodiments, the exemplary microfluidic device also contains a thirdinlet 218. In this example, the third inlet 218 is configured to acceptanother aqueous fluid. This may be the same or different as the aqueousfluid added to the first inlet 201 and/or the second inlet 202. Thethird inlet 218 may be configured to accept beads as provided herein. Inother examples of a microfluidic device, there may only be a singleinlet stream. In some embodiments of a microfluidic device, there arefour or more inlets. In some embodiments the four or more inlets may beaqueous inlets. The exemplary microfluidic device shown in FIG. 11further comprises an inlet 203 for carrier fluid (e.g. an oil immisciblewith an aqueous fluid) in fluid connection with a droplet formationjunction or extrusion junction 204. The inlet 203 in this example isconnected to the droplet formation junction 204 by two channels, eachreaching an aqueous stream channel at the same point on opposite sidesof the aqueous stream channel. The droplet formation junction 204comprises a microfluidic channel that continues down the flow pathtowards cleavage region 206. Near cleavage region 206 is a UV waveguide205 configured to deliver light into the microfluidic channel of thecleavage region 206. In some embodiments, the UV waveguide is afiberoptic wave guide. The UV waveguide 205 is embedded in the plane ofthe device such that the light emitted enters the microfluidic channelof cleavage region 206 from the device plane. In some embodiments, theUV waveguide comprises a parabolic lens at an end closest to thecleavage region. In some embodiments, the parabolic lens is configuredto columnate light inside the cleavage region. In some embodiments, theparabolic lens, or a curved lens, minimizes the tendency for the lightfrom the UV waveguide to be scattered. In some embodiments, the cleavageregion is exposed to UV light projected normal to the circuit plane,exposing a defined area to UV where the compound is cleaved. In someembodiments, an Optical Glue 217 is provided with the UV waveguide. Insome embodiments, the Optical Glue 217 helps to minimize light beingscattered by UV waveguide. Also near cleavage region 206 may be a pillar(not shown) configured to fix a fiberoptic manifold which can beconfigured to emit light from above the plane of the device into themicrofluidic channel of cleavage region 206. The device also comprisesan inlet for calibration fluid 207 a in fluid connection with thecleavage region 206 and an outlet for calibration fluid 207 b. The inletfor calibration fluid 207 a is configured to receive and deliver to thecleavage region 206 a fluid configured to normalize photon exposurewithin the cleavage region. In some embodiments, the cleavage region 206comprises a serpentine flow path. After passing through the cleavageregion 206, the calibration fluid exits through the outlet forcalibration fluid 207 b. The cleavage region 206 is in fluidcommunication via a microfluidic channel to an incubation region 209. Inthe example of FIG. 11, the incubation region 209 contains a series ofwidened chambers, each chamber connected to the next chamber in theseries by a microfluidic channel. The configuration of these chambersaffect the flow rate and residence time of the droplets formed atdroplet formation region 204 through the device. In some embodiments,the chambers are configured to prevent trapping of droplets as they passthrough incubation region 209. Such configuration of the chambers isparticularly important when using a carrier fluid that is denser thanthe aqueous droplets (e.g. 3-ethoxyperfluoro(2-methylhexane)). In someembodiments, the height of the chamber is about 30 μm to about 1,000 μm.In some embodiments, the height of the chamber is about 50 μm to about500 μm. In some embodiments, the depth of the chambers of this exemplarymicrofluidic device (FIG. 11) is larger than the depth of the chambersin the device from FIG. 9A. As such, in some embodiments, this exemplarydevice (FIG. 11) provides for a longer incubation region since a largerdepth would result in faster moving droplets, and thereby a decreasedresidence time if the same length of incubation region as compared tothe device in FIG. 9A was used. In some embodiments, collection chambers219 are optionally provided with this exemplary microfluidic device.Configured on either end of incubation region 209 are bypass shunts 208a and 208 b. The bypass shunts 208 a and 208 b are configured to allow afluid coupled to the shunt to flow in or out of the main microfluidicchannel. If fluid is diverted out of the main microfluidic channel atbypass shunt 208 a, the material will not pass through incubation region209. Positioned downstream of incubation region 209 is inlet for carrierfluid 210. Inlet for carrier fluid 210 is in fluid communication withthe main microfluidic channel of the device and is configured to deliveradditional immiscible carrier fluid into the main microfluidic channelin order to space droplets as desired. Also in fluid communication withthe main microfluidic channel is inlet for carrier fluid 211, which isconfigured to deliver droplet focusing oil into the main microfluidicchannel. In some embodiments, downstream of inlets for carrier fluid 210and 211 is detection position 216. The detection position 216 indicatesthe point on the device that the desired signal from the assay being runon the chip is detected. The detection position 216 may be based on analignment of an objective or fiber that directs an excitation light atthe sample passing detection position 216 and an additional objective orfiber coupled to a detector configured to detect an emission fromdetection position 216. Alternatively, the objective for the excitationlight may be configured to also collect the emission. In someembodiments, the excitation source is reflected from detection position216 through an inverted objective lens, where the emission is collected,columnated, and directed through optical fibers for quantification by aphotomultiplier tube or other detector. In some embodiments, theobjective or fiber aligned at detection position 216 is not coupled tothe device. When not coupled to the device, the detector or emissionobjective or fiber can be moved to adjust the detection positions 216 onthe device in order to adjust the time between detection and sorting.When not coupled to the device, the detector or emission objective mayalso be moved for use in calibration of the device or initiation of thedevice, thus allowing a single light source to be used for multiplefunctions. Downstream of inlets for carrier fluid 210 and 211 anddetection position 216 is discrimination junction electrode 212. Thediscrimination junction electrode 212 may be a dielectrophoresiselectrode configured to propel droplets down outlet 214 if the dropletis determined to display a desired signal or to outlet 215 if thedroplet is determined to lack a desired signal. The discriminationjunction electrode 212 is connected to a discrimination junction groundcircuit, which is connected to the device at circuit connection points213 a and 213 b.

FIG. 16 provides a data set indicating confinement of the UV lightemitted to a cleavage region of a microfluidic device described herein.As such, UV light is not scattered throughout a microfluidic device thatresults in additional encoded effectors from being released while alongan assay flow path. In some embodiments, targeting and confining the UVlight onto a specific region of an assay flow path helps ensure apredetermined amount of encoded effector is released. As an exemplarymethod to confirm such confined UV emission, an assay flow path may bepre-filled with an assay comprising encapsulations having a fluorophoredye. Thus, a number of encapsulations are located downstream of a UVexposure region (e.g., cleavage region), and would not be expected toprovide any detectable signals at a detection point of a microfluidicdevice. As depicted in FIG. 16, an incubation delay period of 1218seconds is shown wherein there are minimal encapsulations exhibitingdetectable signals, followed by a distinct number of encapsulationshaving detectable signals. As such, the UV light emitted was generallyconfined to the cleavage region of a microfluidic, such that theencapsulations passing through the cleavage region was exposed to the UVlight, within minimal scattered light being exposed to encapsulationsfurther along the assay flow path.

FIG. 20 depicts an example of performing fluorescence assay kineticsusing the microfluidic device from FIGS. 9A and 10 is provided. In someembodiments, the fluorescence is measured at various locations withinthe assay flow path, so as to measure the progression of interactionbetween an encoded effector and sample as it is incubated. FIG. 21provides a depiction for positioning a laser spot in a given channelposition so as to measure the PMT emission. FIGS. 23A-24B provide agraphical output of the intensity measured by an assay at differentincubation times (PMT 1). For example, FIGS. 23A-B depicts a raw signaland real-time smoothing intensity measured at the outset of theincubation period (T=0 s), wherein a very low count is measured (e.g.,25 counts at peak). By contrast, FIGS. 24A-B depicts a raw signal andreal-time smoothing intensity (PMT 1) measured at the pre-sort junctionof the incubation period (T=1333 s), wherein a significantly highercount is measured (approximately 380 counts at the peak). FIG. 25provides a comparison of data quality between standard microplate assayand a microfluidic device as described herein. The Figure traces showthe kinetic activity of a protease within a microplate (left) anddroplet compartment (right) on a microfluidic device. The uniformincubation time provides high reproducibility and uniformity at eachtime-point, reducing variance and proving strong statisticalsignificance compared to negative control better than a microplate

Screen Normalization Methods

Provided herein are methods for improving the output results of screensutilizing encoded effectors. Other methods suffer from high rates offalse negatives or positives due to variable loading of effectors orencodings on scaffolds. The variations in amounts of effector orencoding loaded on a scaffold may be due to either low concentration ofencoding/effector on scaffolds, or due to degradation of theencoding/effector during synthesis, the screening process, or storage.In some embodiments, the methods described herein overcome thislimitation by amplifying the level of encodings on scaffolds to uniformlevels. Thus, all the encodings are present at substantially the samelevel and none are drowned out by higher signals from more abundantencodings.

Additionally, in some embodiments, the methods provided herein provide ameans for determining the concentration of effectors bound to scaffolds.In some embodiments, effector loading in an entire library can bedetermined. Having knowledge of the effector load on a scaffold canallow for determination if an effector that displays a positive resultin a screen is due to high potency of the effector of interest, or ifthat particular effector was present at a high concentration within theencapsulation which was screened. Thus, the methods provided herein givethe user a way to readily ascertain how potent a particular effector isand can help remove false positives from an effector screening set.

Further provided herein are methods for amplifying primers for linkingnucleic acids from the samples to the encoding for optimal detection ofnucleic acids. In methods without this amplification step, incompletecapture of nucleic acids released by the sample may occur due to lowlevels of encodings present on the scaffolds. Lower levels of nucleicacid capture could be interpreted as a lack of potency. By amplifyingthe primers within the encapsulation during or after the screen iscompleted, all of the sample nucleic acids can be captured. Thus, themethod improves the readout of nucleic acid levels in a screen. In someinstances, this results in improved yield and knowledge of theexpression levels of various sample components or other knowledgeascertainable from capturing sample nucleic acids.

Barcode Normalization Method

Provided herein are methods for normalization of nucleic acid encodinglevels across a library after performing a screen. During a libraryscreen of nucleic acid encoded effectors, the levels of nucleic acidencodings bound to beads can vary substantially from bead to bead. Thiscan be due to low synthesis yields during synthesis of the bead, or dueto damage to the encoding itself during the screen or during storage.Some beads may have concentrations of encodings bound to beads far inexcess of other beads. Consequently, when sequencing the resulting “hit”beads after performing a screen to determine which effectors wereefficacious, effectors whose encodings are low in concentration aredifficult to detect. This is due to the amplification reactions thatoccur during sequencing, which results in a much higher presence ofencodings whose concentrations start higher. For example, amplifying theencodings from a pooled collection of encoded effectors can generatenoise (e.g., background signal) during sequencing analysis, arising fromtemplate switching, or mis-hybridization, which generate chimericsequences, which are misrepresentative of the true effector population.Therefore, it would take a prohibitively high number of reads to detectencodings which are present in substantially lower concentrations thanothers. For this reason, a method to normalize the levels of nucleicacid encodings after a screen is highly desirable and advantageous, asit allows detection of substantially all effectors that had a positiveresult in the screen. In an exemplary method, isolated amplification ofeach encoding from a collection of encoded effectors helps to preventtemplates from different encodings being formed from the mechanismsleading to chimeric sequences.

In some embodiments, a plurality of screened encoded effectors andcorresponding scaffolds are provided in a plurality of correspondingencapsulations, wherein each scaffold is bound to one or more nucleicacid encodings that encode a corresponding screened encoded effector. Insome embodiments, the plurality of encapsulations are lysed. In someembodiments, contents within the plurality of encapsulations that wereunbound to a scaffold are removed. In some embodiments, the plurality ofscaffolds are then suspended in a liquid medium. In some embodiments,the plurality of scaffolds are then encapsulated in a plurality of newencapsulations, wherein each new encapsulation encapsulates one or morescaffolds. In some embodiments, the nucleic acid encodings of the beads.In some embodiments, the nucleic acids of each bead are amplified toform corresponding amplified nucleic acid encodings. In someembodiments, the amplified nucleic acid encodings within the pluralityof new encapsulations are limited to the nucleic acid encodings andreagents within the respective new encapsulation, thereby improvinguniformity of the number of amplicons representing each encoding. Insome embodiments, the amplified nucleic acid encodings are amplified,such that the concentration of the amplified nucleic acid encodings foreach scaffold are within a minimum level of uniformity to each other.

The nucleic acid encoded library can be subjected to a screen. Any typeof screen can work with the methods and systems provided herein. In someembodiments, the screen previously performed is one of the screeningmethods provided herein. In some embodiments, the screened encodedeffectors have been sorted in the previous screen. In some embodiments,only the “hit” effector beads from the library screen are included inthe present method. In some embodiments, providing the screened encodedeffectors and corresponding scaffolds comprises performing a screen ofthe nucleic acid encoded library. In some embodiments, the screencomprises a sorting step to separate nucleic acid encoded effectors thatdisplayed a positive result in the screen.

In some embodiments, the screened encoded effectors and correspondingscaffolds are provided in an emulsion, within a plurality ofencapsulations. The provided encapsulations containing the screenedencoded effectors and scaffolds may be lysed by a variety of methods. Insome embodiments, lysing the encapsulations comprises introducing ademulsifying reagent, filtration, or sonication to the emulsion. In someembodiments, lysing the encapsulations comprises introducing ademulsifying reagent to the emulsion. In some embodiments, lysing theencapsulations comprises filtering the emulsion. In some embodiments,lysing the encapsulations comprises introducing a demulsifying reagentto the emulsion.

Any demulsifying reagent can be used with the methods and systemsprovided herein. In some embodiments, the demulsifying reagent is anacid or a salt. In some embodiments, the demulsifying reagent is anacid. In some embodiments, the demulsifying reagent is sulfuric acid orhydrochloric acid. In some embodiments, the demulsifying reagent is anorganic acid. In some embodiments, the demulsifying reagent is a salt.In some embodiments, the salt is sodium chloride, potassiumpyrophosphate, or sodium sulfate. In some embodiments, the salt issodium chloride. In some embodiments, the salt is potassiumpyrophosphate. In some embodiments, the salt is sodium sulfate.

In some embodiments, the scaffolds with the encapsulations are washed toremove unbound contents. In some embodiments, washing the scaffoldscomprises rinsing the scaffolds with a wash buffer. In some embodiments,the wash buffer is an aqueous buffer, an organic solution, or a mixturethereof. In some embodiments, the wash buffer is an aqueous buffer. Insome embodiments, the buffer is from pH 4 to pH 10. In some embodiments,the buffer is from pH 5 to 9. In some embodiments, the buffer is from pH6 to pH 8. In some embodiments, the pH is about pH 7. In someembodiments, the wash buffer is a phosphate buffer. In some embodiments,the wash buffer is an isotonic buffer. In some embodiments, the washbuffer is an organic solution. In some embodiments, the organic solutioncomprises methanol, ethanol, isopropyl alcohol, acetonitrile, benzene,toluene, dichloromethane, ethyl acetate, hexanes, any other organicsolvent, or any combination thereof. In some embodiments, the washbuffer comprises a denaturing agent.

Washing the scaffolds may remove unbound content from the scaffoldsand./or that were located within the corresponding encapsulation. Insome embodiments, at least 50%, at least 60%, at least 70%, at least80%, or at least 90% of unbound contents are removed from the scaffoldsduring one or more was steps. In some embodiments, at least 90%, atleast 95%, at least 97%, at least 98%, or at least 99% of unboundcontents are removed from the scaffolds during one or more wash steps.

Multiple washes may be performed. In some embodiments, the scaffolds aresubject to multiple wash and collection steps. In some embodiments, thescaffolds are collected by centrifugation or filtration after each washstep. In some embodiments, the scaffolds are collected by centrifugationafter each wash step. In some embodiments, the scaffolds are collectedby filtration after each wash step. In some embodiments, there is asingle wash step. In some embodiments, there are 2 wash steps. In someembodiments, the was step is repeated 3, 4, 5, 6, 7, 8, 9, 10 or moretimes.

After the wash step, in some embodiments, the scaffolds are suspended ina liquid medium. In some embodiments, the liquid medium is an aqueoussolution. In some embodiments, the liquid medium comprises an organicsolvent. In some embodiments, the liquid medium is compatible withnucleic acid amplification. In some embodiments, the liquid mediumcomprises the amplification mix.

In some embodiments, the scaffolds are then encapsulated in a pluralityof encapsulations (“new encapsulations”). In some embodiments, thescaffolds are encapsulated into a plurality of droplets. In someembodiments, the scaffolds are reintroduced into an emulsion. In someembodiments, each new encapsulation comprises one or more scaffolds. Insome embodiments, the scaffolds are encapsulated such that a majority ofthe new encapsulations comprise one or more single scaffolds. Inembodiments, each droplet comprises an amplification mix.

In some embodiments, encapsulating the scaffolds or re-introducing thescaffold into an emulsion comprises placing the scaffolds through amicrofluidic device. In some embodiments, the microfluidic device is amicrofluidic chip. In some embodiments, the scaffolds are reintroducedinto an emulsion by placing the scaffolds into a one-pot emulsifier.

As described herein, in some embodiments, the scaffold is a solidsupport. In some embodiments, the scaffold is a bead, a fiber,nanofibrous scaffold, a molecular cage, a dendrimer, or a multi-valentmolecular assembly. In some embodiments, the scaffold is a bead. In someembodiments, the bead is a polymer bead, a glass bead, a metal bead, ora magnetic bead. In some embodiments, the bead is a polymer bead. Insome embodiments, the bead is a glass bead. In some embodiments, thebead is a metal bead. In some embodiments, the bead is a magnetic bead.Beads for use in the systems and methods as described herein can be anysize. In some embodiments, the beads are at most 10 nm, at most 100 nm,at most 1 μm, at most 10 μm, or at most 100 μm in diameter. In someembodiments, the beads are at least 10 nm, at least 100 nm, at least 1μm, at least 10 μm, or at least 100 μm in diameter. In some embodiments,the beads are about 10 μm to about 100 μm in diameter.

In some embodiments, the amplification mix can be added to the newencapsulations in a separate step. In some embodiments, theamplification mix is added after the plurality of encapsulations areformed. In some embodiments, the amplification mix is encapsulated atthe same time the scaffolds are being encapsulated. In some embodiments,the amplification mix is added after reintroducing the scaffolds into anemulsion. In some embodiments, the amplification mix is added bypico-injection. In some embodiments, the amplification mix is added bydroplet merging. In some embodiments, the amplification mix is added atthe encapsulation step.

The amplification mix is capable of amplifying the nucleic acids in thenew encapsulations. In some embodiments, the amplification mix comprisesPCR reagents. In some embodiments, the amplification mix comprisesreagents for room temperature amplification.

In some embodiments, the nucleic acid encodings of each scaffold areamplified to form amplified nucleic acid encodings, such that theconcentration of the amplified nucleic acid encodings for each scaffoldare within a minimum level of uniformity to each other. In someembodiments, the minimum level of uniformity comprises a concentrationof nucleic acid encodings in each new encapsulation, wherein about 90%of the new encapsulations have a concentration of amplified nucleic acidencodings within about 10% of an average concentration of amplifiednucleic acid encodings in the plurality of new encapsulations. In someembodiments, the minimum level of uniformity comprises a concentrationof nucleic acid encodings in each new encapsulation, wherein about 80%of the new encapsulations have a concentration of amplified nucleic acidencodings within about 20% of an average concentration of amplifiednucleic acid encodings in the plurality of new encapsulations. In someembodiments, the minimum level of uniformity comprises a concentrationof nucleic acid encodings in each new encapsulation, wherein about 75%of the new encapsulations have a concentration of amplified nucleic acidencodings within 25% of an average concentration of amplified nucleicacid encodings in the plurality of new encapsulations. In someembodiments, the minimum level of uniformity comprises a concentrationof nucleic acid encodings in each new encapsulation, wherein about 70%to about 90% of the new encapsulations have a concentration of amplifiednucleic acid encodings within about 10% to about 30% of an averageconcentration of amplified nucleic acid encodings in the plurality ofnew encapsulations. In some embodiments, the minimum level of uniformitycomprises a concentration of nucleic acid encodings in each newencapsulation, wherein about 70% to about 90% of the new encapsulationscontaining scaffolds have a concentration of amplified nucleic acidencodings within 10-fold, 15-fold, 20-fold, 50-fold, or 100-fold of eachother.

In some embodiments, sequencing the amplified nucleic acid encodingsresults in lower background signal than a nucleic acid encoded librarythat has not been subjected to the method. In some embodiments, thebackground signal is reduced by at least 10%, at least 20%, at least30%, at least 40%, or at least 50%. In some embodiments, the backgroundsignal is reduced by at least 75%. In some embodiments, the backgroundsignal is reduced by at least 90%. In some embodiments, the backgroundsignal is reduced by at least 95%.

In some embodiments, the lower background signal allows for detection ofnucleic acid encoded effectors whose encoding concentrations before thescreen are 100×, 1000×, 10000×, 100000×, or 1000000× lower inconcentration than the average encoding concentration in the library. Insome embodiments, the lower background signal allows for detection ofnucleic acid encoded effectors whose encoding concentrations before thescreen are 100× lower in concentration than the average encodingconcentration in the library. In some embodiments, the lower backgroundsignal allows for detection of nucleic acid encoded effectors whoseencoding concentrations before the screen are 1000× lower inconcentration than the average encoding concentration in the library. Insome embodiments, the lower background signal allows for detection ofnucleic acid encoded effectors whose encoding concentrations before thescreen are 10000× lower in concentration than the average encodingconcentration in the library. In some embodiments, the lower backgroundsignal allows for detection of nucleic acid encoded effectors whoseencoding concentrations before the screen are 100000× lower inconcentration than the average encoding concentration in the library. Insome embodiments, the lower background signal allows for detection ofnucleic acid encoded effectors whose encoding concentrations before thescreen are 1000000× lower in concentration than the average encodingconcentration in the library.

Primer Amplification Method

Provided herein is a method for amplifying a primer to maximize cellularnucleic acid capture. In some screening methods provided herein, nucleicacid contents of cells are transferred to the nucleic acid encodings ofvarious effectors. The nucleic acid encodings are sometimes linked toscaffolds, such as beads. However, a library of beads may compriseindividual beads that may have dramatically different levels of nucleicacids encodings on the beads. Consequently, such beads are unable toattach significant levels of cellular nucleic acids, or other beads areable to attach substantially more levels of cellular nucleic acids. Suchdiscrepancies make it difficult to determine if the cellular nucleicacid level differences are due to the differential effects of variouseffectors, or if there were simply less capture sites available togather the cellular nucleic acids. Therefore, a method of producingadditional primers to label the cellular nucleic acids with the nucleicacid encoding would have substantial benefits.

In one aspect, provided herein, is a method for amplifying a primer tomaximize cellular nucleic acid capture. In some embodiments, the primeris a copy of a nucleic acid encoding (encoded nucleic acid primer). Insome embodiments, the method comprises encapsulating a nucleic acidencoded scaffold with one or more cells, an amplification mix, and anicking enzyme. In some embodiments, the nicking enzyme targets aspecific nucleotide sequence. As described herein, a nucleic acidencoded scaffold is bound to one or more nucleic acid encodings. In someembodiments, the one or more nucleic acid encodings comprise a specificnucleotide sequence. In some embodiments, the cell is lysed to releaseone or more cellular nucleic acids. In some embodiments, the nucleicacid encoding is nicked with the nicking enzyme, thereby creating anencoded nucleic acid primer. In some embodiments, nicking refers to asingle strand of a an encoding being displaced. In some embodiments, thenicking enzyme targets a specific site in the nucleic acid encoding. Insome embodiments, the specific site comprises the specific nucleotidesequence. In some embodiments, nicking the nucleic acid encoding createsan encoded nucleic acid primer. In some embodiments, the encoded nucleicacid primer is amplified. In some embodiments, the encoded nucleic acidprimer is amplified via interaction between the nicking site and theamplification mix. In some embodiments, a released cellular nucleic acidis labeled with an encoded nucleic acid primer.

In some embodiments, amplifying the encoded nucleic acid primercomprises first creating a copy of the nucleic acid encoding, which isextended from the nicking site, followed by nicking the nucleic acidencoding copy. In some embodiments, amplifying the encoded nucleic acidprimer comprises simultaneously 1) creating a copy of the nucleic acidencoding, which extends from the nicking site, and 2) displacing thenucleic acid encoding copy.

In some embodiments, the amplification mix comprises an amplificationenzyme. In some embodiments, the amplification enzyme enables for thecreation of a nucleic acid encoding copy, and then the subsequentnicking. In some embodiments, the nicking enzyme enables the nicking ofthe copy of the nucleic acid encoding copy. In some embodiments, theamplification enzyme enables for a copy of the nucleic acid encoding tobe simultaneously created and displaced. In some embodiments, theamplification enzyme is a polymerase. In some embodiments, the creationof nucleic acid encoding copies and nicking, or the simultaneouscreation and displacement of the nucleic acid encoding copies, repeatsto generate a population of single stranded nucleic acid encodings thatserve as a primer (encoded nucleic acid primer) for labeling cellularnucleic acids. In some embodiments, the encoded nucleic acid primers aregenerated isothermally.

In some embodiments, each encoded nucleic acid primer comprises acapture site that prescribes a target cellular nucleic acid to label aspecific released cellular nucleic acid. In some embodiments, the targetnucleic acid is a target mRNA. In some embodiments, the target mRNAencodes a protein of interest. In some embodiments, the nicking enzymeenables an increase in target mRNA capture and labeling with the nucleicacid encoding. In some embodiments, the target mRNA capture is increasedby at least 10%, 25%, 50%, 100%, or 200%.

In some embodiments, a plurality of cellular nucleic acids are labeledwith an respective encoded nucleic acid primer. In some embodiments, thenucleic acid encoded scaffold comprises a bead, and the encoded nucleicacid primer comprises a unique bead barcode and an effector encoding.

FIG. 3 illustrates an exemplary method for amplifying a primer tomaximize cellular nucleic acid capture, as described herein. As shown inFIG. 3, in step 1, a nucleic acid encoded scaffold is shown with thenucleic acid encoding bound thereto, wherein a plurality of cellularencodings (e.g., nucleic acid) are also shown to have been released froma lysed cell. In some embodiments, the nucleic acid encoded scaffold andcellular encodings are provided within an encapsulation. The nickingsite is identified on the nucleic acid encoding, along with a capturesite. In some embodiments, the nicking site corresponds to a specificnucleotide sequence in the nucleic acid encoding. As shown in step 2,the nucleic acid encoding is nicked at the nicking site. As shown, insome embodiments, nicking herein refers to a single strand of theencoding being displaced from the nucleic acid encoded scaffold. Asshown in steps 3-4 of FIG. 3, an amplification enzyme may interact withthe nicking site, thereby creating a new copy of the nucleic acidencoding (step 4), while the previously nicked nucleic acid encodingcopy (encoded nucleic acid primer) is unbound and moves within theencapsulation, such that the encoded nucleic acid primer may interactwith a released cellular encoding (e.g., cellular nucleic acid), asshown in step 5. In some embodiments, the encoded nucleic acid primerlabels the cellular encoding. In some embodiments, the capture site ofthe encoded nucleic acid primer prescribes a targeted cellular nucleicacid. In some embodiments, an enzyme enables such labeling. As shown instep 6, the encoded cell encoding is labeled with the encoded nucleicacid primer, while a created copy of the nucleic acid encoding isdisplaced from the scaffold, wherein the process returns to step 3.

The cell may be lysed in order to release the desired nucleic acids andto make the desired nucleic acids available for amplification. In someembodiments, the encapsulation further comprises a cell lysis buffer. Insome embodiments, the lysis buffer is added by pico-injection. In someembodiments, the lysis buffer comprises a salt. In some embodiments, thelysis buffer comprises a detergent. In some embodiments, the detergentis SDS, Triton, or Tween. In some embodiments, the lysis buffercomprises a chemical which causes cell lysis. In some embodiments, celllysis buffer is added to the encapsulation. In some embodiments, thecell lysis buffer is added to the encapsulation by pico-injection.

In some embodiments, the encapsulation is a droplet, an emulsion, amacrowell, a microwell, a bubble, or a microfluidic confinement. Once anencapsulation is formed, any component inside the encapsulation mayremain in the encapsulation until the encapsulation is destroyed orbroken down. In some embodiments, the encapsulations used in hereinremain stable for at least 4 hours, at least 12 hours, at least 1 day,at least 2 days, at least 3 days, or at least 1 week. In someembodiments, the encapsulations are stable for the duration of thescreen to be performed so that no intermingling of reagents betweenencapsulations occurs.

In some embodiments, the encapsulation is a droplet. In someembodiments, the droplet is at most 1 picoliter, at most 10 picoliters,at most 100 picoliters, at most 1 nanoliter, at most 10 nanoliters, atmost 100 nanoliters, or at most 1 microliter in volume. In someembodiments, the droplet is at least 1 picoliter, at least 10picoliters, at least 100 picoliters, at least 1 nanoliter, at least 10nanoliters, at least 100 nanoliters, or at least 1 microliter in volume.In some embodiments, the droplet is between about 200 picoliters andabout 10 nanoliters.

In some embodiments, the droplet is an aqueous droplet in a larger bodyof oil. In some embodiments, the oil acts as an immiscible carrierfluid. In some embodiments, the droplets are placed in an oil emulsion.In some embodiments, the oil comprises a silicone oil, a fluorosiliconeoil, a hydrocarbon oil, a mineral oil, a paraffin oil, a halogenatedoil, a fluorocarbon oil or any combination thereof. In some embodiments,the oil comprises a silicone oil. In some embodiments, the oil comprisesa fluorosilicone oil. In some embodiments, the oil comprises ahydrocarbon oil. In some embodiments, the oil comprises a mineral oil.In some embodiments, the oil comprises a paraffin oil. In someembodiments, the oil comprises a halogenated oil. In some embodiments,the oil is a fluorocarbon oil.

In some embodiments, an amplification mix is used to amplify nucleicacid encodings to create additional primers for labeling cellularnucleic acids of interest in a screen. In some embodiments, theamplification mix is an isothermal amplification mix. In someembodiments, the isothermal amplification mix comprises reagents forloop-mediated isothermal amplification (LAMP), strand displacementamplification (SDA), helicase-dependent amplification (HAD), recombinasepolymerase amplification (RPA), rolling circle replication (RCA), ornicking enzyme amplification reaction (NEAR). In some embodiments, theencapsulation further comprises reagents for isothermal amplification ofthe target nucleic acid. In some embodiments, the method comprisesadding reagents for isothermal amplification to the encapsulation. Insome embodiments, the reagents for isothermal amplification are targetedto the specific nucleic acid sequence. In some embodiments, theamplification mix comprises a nicking enzyme. In some embodiments, theamplification mix comprises a nicking-enzyme amplification mixture. Insome embodiments, the nicking enzyme is an endonuclease. In someembodiments, the nicking enzyme is a restriction enzyme. In someembodiments, the amplification mix comprises a reverse transcriptase. Insome embodiments, the amplification mix comprises an amplificationenzyme. In some embodiments, the amplification enzyme comprises apolymerase.

In some embodiments, the specific nucleotide sequence of interest can beamplified within the encapsulation. In some embodiments, the methodcomprises amplifying the cellular nucleic acid comprising the specificnucleotide sequence to produce amplified cellular nucleic acids. In someembodiments, amplifying the cellular nucleic acids is accomplished byPCR. In some embodiments, amplifying the cellular nucleic acids isaccomplished by isothermal amplification. In some embodiments, cellularnucleic acids comprising the specific nucleotide sequence are amplified.In some embodiments, the amplified cellular nucleic acid is barcodedwith the nucleic acid encoding the scaffold.

Any type of scaffold may be utilized in this method. In someembodiments, the scaffold acts as a solid support and keeps the nucleicacid encoding the scaffold linked in space to the scaffold. In someembodiments, the scaffold is a structure with a plurality of attachmentpoints that allow linkage of one or more molecules. In some embodiments,the nucleic acid encoding the scaffold is bound to the scaffold. In someembodiments, the scaffold is a solid support. In some embodiments, thescaffold is a bead, a fiber, nanofibrous scaffold, a molecular cage, adendrimer, or a multi-valent molecular assembly.

In some embodiments, the scaffold is a bead. In some embodiments, thebead is a polymer bead, a glass bead, a metal bead, or a magnetic bead.In some embodiments, the bead is a polymer bead. In some embodiments,the bead is a glass bead. In some embodiments, the bead is a metal bead.In some embodiments, the bead is a magnetic bead.

Beads for use in the systems and methods as described herein can be anysize. In some embodiments, the beads are at most 10 nm, at most 100 nm,at most 1 μm, at most 10 μm, or at most 100 μm in diameter. In someembodiments, the beads are at least 10 nm, at least 100 nm, at least 1μm, at least 10 μm, or at least 100 μm in diameter. In some embodiments,the beads are about 10 μm to about 100 μm in diameter.

The scaffolds may comprise effectors attached to the scaffold. In someembodiments, the effectors are attached to the scaffold by the cleavablelinkers described herein. In some embodiments, the cleavable linker iscleaved by electromagnetic radiation, an enzyme, chemical reagent, heat,pH adjustment, sound or electrochemical reactivity. In some embodiments,the cleavable linker is cleaved from the scaffold using electromagneticradiation. In some embodiments, the amount of effector cleaved iscontrolled by the intensity or duration of exposure to electromagneticradiation. In some embodiments, the cleavable linker is cleaved using acleavage reagent. In some embodiments, the amount of effector cleaved iscontrolled by the concentration of the cleavage reagent in theencapsulation. In some embodiments, the effector is cleaved from thescaffold using an enzyme. In some embodiments, the enzyme is a protease,a nuclease, or a hydrolase. In some embodiments, the rate of effectorcleavage is controlled by the amount of enzyme in the encapsulation.

In some embodiments, the encoded nucleic acid primers amplified in thepresent methods are utilized to detect and quantify the amount of atarget nucleic acid in the one or more cells being screened with aneffector utilizing the nucleic acid encoded scaffold. In someembodiments, the encoded nucleic acid primer hybridizes with a targetnucleic acid.

In some embodiments, the specific nucleotide sequence acts as anamplification primer with the target nucleic acid. In some embodiments,the target nucleic acid is barcoded with the nucleic acid encoding thescaffold using the specific nucleotide sequence. In some embodiments,the target nucleic acid is barcoded with the nucleic acid encoding thescaffold using the specific nucleotide sequence which has been extendedwith the nucleic acid encoding the scaffold.

The target nucleic acid can by any type of nucleic acid from a cell. Insome embodiments, the target nucleic acid is a target mRNA. In someembodiments, the target mRNA encodes a protein of interest. In someembodiments, the target nucleic acid comprises a plurality of targetmRNAs. In some embodiments, barcoding the plurality of target mRNAscreates an expression fingerprint of the cell treated with an effector.In some embodiments, the target nucleic acid is genomic DNA. In someembodiments, the target nucleic acid is mitochondrial DNA.

The methods provided herein increase target nucleic acid capture andlabeling with the nucleic acid encoding the scaffold. In someembodiments, target nucleic acid capture is increased by at least 10%,25%, 50%, 100%, or 200% compared to a method without the nicking enzymethat targets the specific nucleotide sequence. In some embodiments,target nucleic acid labeling is increased by at least 10%, 25%, 50%,100%, or 200% compared to a method without the nicking enzyme thattargets the specific nucleotide sequence. In some embodiments, targetnucleic acid capture is increased by at least 5-fold, at least 10-fold,at least 50-fold, or at least 100-fold compared to a method without thenicking enzyme that targets the specific nucleotide sequence. In someembodiments, target nucleic acid barcoding is increased by at least5-fold, at least 10-fold, at least 50-fold, or at least 100-foldcompared to a method without the nicking enzyme that targets thespecific nucleotide sequence.

In some embodiments, labeling the cellular nucleic acids with encodednucleic acid primers, as described herein, comprises barcoding thecellular nucleic acids. The encapsulation can further comprise barcodingreagents. In some embodiments, the encapsulation further comprisesbarcoding reagents. In some embodiments, the encapsulation furthercomprises barcoding reagents to effectuate the barcoding of the cellularnucleic acids with the encoded nucleic acid primers. In someembodiments, the encapsulation further comprises barcoding reagents toeffectuate the barcoding of the nucleic acid encoding the scaffold withamplified nucleic acids.

The barcoding reagents can be any set of reagents that allow the joiningof different nucleic acids. In some embodiments, the barcoding reagentscomprise an enzyme or chemical cross-linking reagent. In someembodiments, the enzyme is a polymerase, a ligase, a restriction enzyme,or a recombinase. In some embodiments, the enzyme is a polymerase. Insome embodiments, the additional reagents comprise a chemicalcross-linking reagent. In some embodiments, the chemical cross-linkingreagent is psoralen.

The amplification of primers described herein can be performed at anytime. In some embodiments, the above methods can be performed at thesame time as an effector screen. In some embodiments, the cell is beingscreened against the effector. In some embodiments, an effector screenoccurs concomitantly with the primer amplification method. In someembodiments, the primer amplification method described herein occursprior to an effector screen. In some embodiments, the method is used toprepare the nucleic acid encoded scaffold for a screen. In someembodiments, the cell is used to prepare the nucleic acid encodedscaffold for a screen.

Effector Load Normalization Method

Provided herein are methods of measuring effector loading onto scaffoldsand libraries of scaffolds. Generally, when a library of encodedeffectors bound to scaffolds is prepared, the final concentration ofeffectors bound to the scaffolds varies considerably among individualscaffolds. This is due to differences in yield of each synthesis step ofthe effector built onto the scaffold. Consequently, when ultimately usedin a screen, different samples may receive different dosages ofeffectors. This can skew the results of the screen, as low potency, highabundance effectors may drown out the signal of higher potency, lowabundance effectors. Thus, a method of determining effector loading ontoscaffolds in a library can help avoid this skewing of results.

Provided herein are methods of measuring effector loading on scaffolds.In some embodiments, the method comprises (a) attaching an effectorsubunit to effector attachment sites on a plurality of scaffolds. Insome embodiments, the method comprises (b) attaching a detectable labelto any remaining free effector attachment sites on the plurality ofscaffolds after the step of attaching an effector subunit. In someembodiments, the method comprises (c) removing a subset of scaffoldsfrom the plurality. In some embodiments, the method comprises (d)measuring the amount of detectable label attached to the subset ofscaffolds to determine the amount of effector subunits successfullyattached to the effector attachment sites. In some embodiments, themethod comprises (e) optionally activating the attached effectorsubunits to create new effector attachment sites. In some embodiments,the listed steps are repeated until a desired effector is assembled. Insome embodiments, the scaffold further comprises a nucleic acid encodingthe effector. In some embodiments, the method further comprisesattaching nucleic acid encoding subunits to the scaffold correspondingto the effector subunits as the effector subunits are added to thescaffold. In some embodiments, there is no activating step after thelast effector subunit is attached.

In some embodiments, each effector subunit attached to the scaffold isindependently an amino acid, a small molecule fragment, a nucleotide, ora compound. In some embodiments, each effector subunit attached to thescaffold is an amino acid. In some embodiments, each effector subunitattached to the scaffold is a compound. In some embodiments, eacheffector subunit attached to the scaffold is a small molecule fragment.In some embodiments, each effector subunit attached to the scaffold is anucleotide.

The effector attachment sites may have any group capable of performing achemical reaction. In some embodiments, the effector attachment sitescomprise reactive functionalities. In some embodiments, the effectorattachment sites comprise amino groups, carboxylate groups, alcoholgroups, phenol groups, alkyne groups, aldehyde groups, or ketone groups.In some embodiments, the effector attachment sites comprise amino orcarboxylate groups. IN some embodiments, the effector attachment sitescomprise biorthogonal or CLICK chemistry reactive groups.

The encoding subunits can comprise functional groups that may react withthe reactive functionalities on the effector attachment site. In someembodiments, the encoding subunits form a covalent bond with thereactive functionalities. In some embodiments, the effector subunitscomprise reactive groups complementary to the effector attachment sites.

The detectable labels, in some embodiments, comprise functional groupsthat may react with the reactive functionalities on the effectorattachment site. In some embodiments, the detectable labels form acovalent bond with the reactive functionalities. In some embodiments,the detectable labels comprise reactive groups complementary to theeffector attachment sites.

The detectable label may any label that can produce a signal that can bedetected and quantified. In some embodiments, the detectable label is afluorophore.

In some embodiments, there is a yield associated with each effectorattachment step. In some embodiments, the yield is measured a percentageof free effector attachment sites after the step of attaching aneffector subunit. In some embodiments, at most 10%, at most 20%, at most30%, at most 40%, or at most 50% of the effector attachment sites arefree after the step of attaching the effector subunit.

A subset of beads may be removed in order to quantify the loading ateach step of the synthesis of the desired effector. In some embodiments,removing a subset of the plurality of scaffolds comprises removing nomore than 1%, no more than 2%, no more than 3%, no more than 5%, or nomore than 10% of the remaining scaffolds. In some embodiments, removinga subset of the plurality of scaffolds comprises removing no more than1% of the remaining scaffolds. In some embodiments, removing a subset ofthe plurality of scaffolds comprises removing no more than 2% of theremaining scaffolds. In some embodiments, removing a subset of theplurality of scaffolds comprises removing no more than 3% of theremaining scaffolds. In some embodiments, removing a subset of theplurality of scaffolds comprises removing no more than 5% of theremaining scaffolds. In some embodiments, removing a subset of theplurality of scaffolds comprises removing no more than 10% of theremaining scaffolds.

In some embodiments, wherein measuring the amount of detectable labelattached to the subset of scaffolds to determine the amount of effectorsubunits successfully attached to the effector attachment sitescomprises comparing the measurement of the detectable label to themeasurement of detectable label on a scaffold without any effectorsubunits attached. In some embodiments, the amount of effector subunitssuccessfully attached to the subset of scaffolds is expressed as apercentage of total attachment sites occupied by the effector subunits.

To begin a new step of attaching effector subunits, a previouslyattached effector subunit may need to be activated. In some embodiments,activation reveals the presence of a new effector attachment site. Insome embodiments, optionally activating the attached effector subunitsto create a new effector attachment site comprises removing a protectinggroup from the attached effector subunit. In some embodiments, theprotecting group is an amino protecting group, a carboxylate protectinggroup, an alcohol protecting group, a phenol protecting group, an alkyneprotecting group, an aldehyde protecting group, or a ketone protectinggroup. In some embodiments, the protecting group is an amino protectinggroup. In some embodiments, the amino protecting group is9-fluorenylmethyloxcarbonyl (Fmoc), tert-butyloxycarbonyl (BOC),carbobenzyloxy (Cbz), benzyl (Bz), tosyl (Ts) or trichloroethylchloroformate (Troc). In some embodiments, the protecting group is acarboxylate protecting group. In some embodiments, the carboxylateprotecting group is a methyl ester, a benzyl ester, a tert-butyl ester,a 2,6-disubstituted phenolic ester, a silyl ester, or an orthoester. Insome embodiments, the protecting group is an alcohol protecting group.In some embodiments, the protecting group is a phenol protecting group.In some embodiments, the protecting group is an alkyne protecting group.In some embodiments, the protecting group is an aldehyde protectinggroup. In some embodiments, the protecting group is a ketone protectinggroup.

The new effector attachment site can be any suitable reactivefunctionality. In some embodiments, the new effector attachment site isthe same functionality as the previous effector attachment site. In someembodiments, the new effector attachment site is a differentfunctionality from the previous effector attachment site.

The desired effectors can be synthesized using any number of steps anduse any number of effector subunits. In some embodiments, steps (a)-(e)are repeated at least 2, at least 3, at least 4, at least 5, at least 6,at least 7, at least 10, or at least 20 times. In some embodiments, thedesired effector is comprised of at least 2, at least 3, at least 4, atleast 5, at least 6, at least 7, at least 10, or at least 20 subunits.

Any type of scaffold may be used with the methods and systems providedherein. In some embodiments, the scaffold is a bead, a fiber, ananofibrous scaffold, a molecular cage, a dendrimer, or a multi-valentmolecular assembly. In some embodiments, the scaffold is a bead. In someembodiments, the bead is a polymer bead, a glass bead, a metal bead, ora magnetic bead. In some embodiments, the bead is a polymer bead. Insome embodiments, the bead is a glass bead. In some embodiments, thebead is a metal bead. In some embodiments, the bead is a magnetic bead.

The beads utilized in the methods provided herein may be made of anymaterial. In some embodiments, the bead is a polymer bead. In someembodiments, the bead comprises a polystyrene core. In some embodiments,the beads are derivatized with polyethylene glycol. In some embodiments,the beads are grafted with polyethylene glycol. In some embodiments, thepolyethylene glycol contains reactive groups for the attachment of otherfunctionalities, such as effectors or encodings. In some embodiments,the reactive group is an amino or carboxylate group. In someembodiments, the reactive group is at the terminal end of thepolyethylene glycol chain. In some embodiments, the bead is a TentaGel®bead.

The polyethylene glycol (PEG) attached to the beads may be any size. Insome embodiments, the PEG is up to 20 kDa. In some embodiments, the PEGis up to 5 kDa. In some embodiments, the PEG is about 3 kDa. In someembodiments, the PEG is about 2 to 3 kDa.

In some embodiments, the PEG group is attached to the bead by an alkyllinkage. In some embodiments, the PEG group is attached to a polystyrenebead by an alkyl linkage. In some embodiments, the bead is a TentaGel® Mresin.

In some embodiments, the bead comprises a PEG attached to a bead throughan alkyl linkage and the bead comprises two bifunction species. In someembodiments, the beads comprise surface modification on the outersurface of the beads that are orthogonally protected to reactive sitesin the internal section of the beads. In some embodiments the beadscomprise both cleavable and non-cleavable ligands. In some embodiments,the bead is a TentaGel® B resin.

Beads for use in the systems and methods as described herein can be anysize. In some embodiments, the beads are at most 10 nm, at most 100 nm,at most 1 μm, at most 10 μm, or at most 100 μm in diameter. In someembodiments, the beads are at least 10 nm, at least 100 nm, at least 1μm, at least 10 μm, or at least 100 μm in diameter. In some embodiments,the beads are about 10 μm to about 100 μm in diameter.

Nucleic acids encoding the effector are utilized in the describedmethod. The nucleic acids encoding the effector may be bound to thescaffold as a pre-synthesized nucleic acid, synthesized concomitantlywith the effector, or synthesized on the scaffold prior to synthesis ofthe effector. In some embodiments, a nucleic acid encoding the effectoris attached to the scaffold. In some embodiments, the method furthercomprises attaching nucleic acid encoding subunits to the scaffoldcorresponding to the effector subunits as the effector subunits areadded to the scaffold.

The methods described herein are especially useful when applied tolibraries of effectors on scaffolds. In some embodiments, libraries ofeffectors are synthesized in parallel. In some embodiments, libraries ofeffectors are synthesized in individual wells. In some embodiments,libraries of effectors are synthesized using high-throughput synthesistechniques. In some embodiments, a library of effector loaded scaffoldsare synthesized concurrently. The library of effector loaded scaffoldscan be any size. In some embodiments, the library comprises at least10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵,or 10¹⁶ effector loaded scaffolds. In some embodiments, each effectorloaded scaffold comprises a unique effector. In some embodiments, someeffector loaded scaffolds are repeated in the library.

In some embodiments, subsets of beads from an effector attachment stepfrom the library are pooled prior to detection of the detectable label.In some embodiments, subsets of beads from all scaffolds in the libraryare pooled together. In some embodiments, a portion of the subset ofbeads from the scaffolds in the library are pooled together.

The pooled subsets of beads are placed into encapsulations for furtheranalysis. An encapsulation refers to the formation of a compartmentwithin a larger system. In some embodiments, the encapsulation is adroplet, an emulsion, a macrowell, a microwell, a bubble, or amicrofluidic confinement. In some embodiments, a majority of theencapsulations comprise a single scaffold.

In some embodiments, the encapsulation is a droplet. In someembodiments, the droplet is at most 1 picoliter, at most 10 picoliters,at most 100 picoliters, at most 1 nanoliter, at most 10 nanoliters, atmost 100 nanoliters, or at most 1 microliter in volume. In someembodiments, the droplet is at least 1 picoliter, at least 10picoliters, at least 100 picoliters, at least 1 nanoliter, at least 10nanoliters, at least 100 nanoliters, or at least 1 microliter in volume.In some embodiments, the droplet is between about 200 picoliters andabout 10 nanoliters.

In some embodiments, the droplet is an aqueous droplet in a larger bodyof oil. In some embodiments, the droplets are placed in an oil emulsion.In some embodiments, the oil comprises a silicone oil, a fluorosiliconeoil, a hydrocarbon oil, a mineral oil, a paraffin oil, a halogenatedoil, or any combination thereof. In some embodiments, the oil comprisesa silicone oil. In some embodiments, the oil comprises a fluorosiliconeoil. In some embodiments, the oil comprises a hydrocarbon oil. In someembodiments, the oil comprises a mineral oil. In some embodiments, theoil comprises a paraffin oil. In some embodiments, the oil comprises ahalogenated oil.

After the scaffolds are placed into encapsulations, the level offluorophore bound to the scaffolds may be assessed. In some embodiments,scaffolds from the subset of scaffolds are binned according to theamount of detectable label detected. In some embodiments, each bincomprises a unique range of detectable label detected. In someembodiments, the bins correspond to 0-25%, 25-50%, 50-75%, and 75-100%loading of detectable label detected compared to scaffolds where noeffector subunit was loaded. In some embodiments, the bins correspond to0-20%, 20-40%, 40-60%, 60-80%, and 80-100% loading of detectable labeldetected compared to scaffolds where no effector subunit was loaded. Insome embodiments, the bins correspond to 0-10%, 10-20%, 20-30%, 30-40%,40-50%, 50-60%, 60-70%, 70-80%, 80-90%, and 90-100% loading ofdetectable label detected compared to scaffolds where no effectorsubunit was loaded. Any combination of bins is acceptable to use withthe methods and systems provided herein.

The bins may then be sequenced to reveal which effectors had particularyields in the attachment step. In some embodiments, the method furthercomprises the step of sequencing encoding nucleic acids or encodingnucleic acid subunits of the pools to reveal which effector subunitscorrespond to a particular yield in a step of attaching effectorsubunits to effector attachment sites. In some embodiments, thesequencing step is performed each time steps (a)-(e) are repeated. Insome embodiments, yields of each step (a)-(e) for each unique scaffoldare collected to create a dataset which reveals the loading of thecomplete desired effector on each scaffold. In some embodiments, yieldsof attachment of each encoder subunit for each unique scaffold arecollected to create a dataset which reveals the loading of the completedesired effector on each scaffold. In some embodiments, the loading ofdesired effector on each unique scaffold is calculated.

Screening Devices and Methods of Use

Further provided herein are devices for use in screening encodedeffectors and methods of use. In some embodiments, the devices providedherein lock an encoded effector into a location. In some embodiments,the sample being screened is similarly fixed in a position. By lockingthe two in place, the risk of encapsulations breaking down or mergingwith other encapsulations may be minimized. In some embodiments, theneed for encapsulation is eliminated entirely. Additionally, knowledgeof the structure of the effector at particular locations of the devicemay allow a user to easily determine which effectors had a desiredeffect on a sample. The devices described below are compatible with anyof the methods described elsewhere herein.

Nucleic Acid Patch Array

Provided herein is an array for screening encoded beads. The array cancomprise nucleic acid patches interspersed on a hydrophobic surface. Thepositioning of the nucleic acid patches on the hydrophobic patch can besuch that when a liquid media is added to the device, droplets formencapsulating the nucleic acid patches, but the hydrophobic surfacesremain free of media. In some embodiments, each nucleic acid patch isencapsulated in its own droplet. In some embodiments, there is no liquidor fluid connection between the different nucleic acid patches after themedia is added. The nucleic acid patches may be able to bind beads,cells, or both. Additionally, the array may further comprise channelsbeneath the surface. The channels can have terminal ends that allow forfluids to flow through the channels to the nucleic acid patches. Suchchannels can allow for the addition of reagents to the nucleic acidpatches.

In one aspect, provided herein, is an array device for screening encodedbeads. In some embodiments, the device comprises a hydrophobic surface.In some embodiments, the device comprises nucleic acid patches. In someembodiments, the nucleic acid patches are interspersed on thehydrophobic surface. In some embodiments, the hydrophobic surface andnucleic acid patches are configured such that when a proscribed amountof media is deployed across the surface each nucleic acid patch iscovered with media. In some embodiments, the hydrophobic surface betweenthe nucleic acid patches does not contain media.

The array device may comprise channels. In some embodiments, the devicecomprises one or more channels beneath the hydrophobic surface. In someembodiments, the channels from a network. In some embodiments, thechannels are microfluidic channels. In some embodiments, the channelsare branched. In some embodiments, the channels comprise terminal endswithin nucleic acid patches. In some embodiments, the channels compriseterminal ends within each nucleic acid patch of the array.

The channels may be configured to deliver liquid solutions to thenucleic acid patches. In some embodiments, the channels are configuredto deliver reagents to the nucleic acid patches. In some embodiments,the reagents are delivered as a liquid solution. In some embodiments,the liquid solution is an aqueous solution.

The channels may be any size. In some embodiments, the channels have adiameter of about 0.1 μm, about 0.5 μm, about 1 μm, about 5 μm, about 10μm, or about 20 μm. In some embodiments, the channels have a diameter ofup to about 0.1 μm, up to about 0.5 μm, up to about 1 μm, up to about 5μm, up to about 10 μm, or up to about 20 μm. In some embodiments, thechannels have a diameter of at least about 0.1 μm, at least about 0.5μm, at least about 1 μm, at least about 5 μm, at least about 10 μm, orat least about 20 μm.

The hydrophobic surface may be made of any suitable hydrophobicmaterial. In some embodiments, the hydrophobic surface is comprised of ahydrophobic polymer. In some embodiments, the hydrophobic surfacecomprises a hydrophobic polymer. In some embodiments, the hydrophobicpolymer comprises a polyacrylic, a polyamide, a polycarbonate, apolydiene, a polyester, a polyether, a polyfluorocarbon, a polyolefin, apolystyrene, a polyvinyl acetal, a polyvinyl chloride, a polyvinylester, a polyvinyl ether, a polyvinyl ketone, a polyvinyl pyridine, apolyvinylpyrrolidone, a polysilane, a polyfluorosilane, a polyperfluorosilane or a combination thereof. In some embodiments, thehydrophobic polymer comprises a polyfluorocarbon. In some embodiments,the hydrophobic polymer comprises a polyperfluorocarbon. In someembodiments, the hydrophobic polymer is fluorinated.

The hydrophobic surface may be a surface functionalized with groupshaving hydrophobic properties. In some embodiments, the hydrophobicsurface is a surface functionalized with hydrophobic groups. In someembodiments, the hydrophobic groups are fatty acids, alkyl groups,alkoxy groups, aromatic groups, alkyl silanes, fluorosilanes,perfluorosilanes, or combinations thereof. In some embodiments, thehydrophobic groups are perfluorosilanes. In some embodiments, thehydrophobic groups are fatty acids. In some embodiments, the hydrophobicgroups are fluorinated fatty acids. In some embodiments, the hydrophobicgroups are perfluorinated fatty acids. In some embodiments, thehydrophobic groups are fluorinated.

The hydrophobic surface may exhibit desired binding properties. In someembodiments, cells do not bind to the hydrophobic surface. In someembodiments, cells do not grow on the hydrophobic surface.

The nucleic acid patches may exhibit desired binding properties. In someembodiments, the nucleic acid patches bind cells. In some embodiments,the nucleic acid patches bind cells through non-specific interaction. Insome embodiments, the nucleic acid patches bind cells through specificinteraction. In some embodiments, the nucleic acid patches areconfigured to attract media. In some embodiments, single nucleic acidpatches encapsulated within single droplets of the media. In someembodiments, the nucleic acid patches are capable of binding beads. Insome embodiments, the beads are nucleic acid encoded beads. In someembodiments, the nucleic acid patches bind beads. In some embodiments,the nucleic acid patches comprise nucleic acids capable of bindingnucleic acid encoded beads. In some embodiments, the nucleic acids bindbeads non-specifically, by binding a complementary nucleic acid on thebead, or by binding another group on the bead. In some embodiments, thenucleic acids bind nucleic acid encoded beads non-specifically, bybinding a complementary nucleic acid on the bead, or by binding anothergroup on the bead.

The nucleic acid patches may comprise any type of nucleic acid. In someembodiments, the nucleic acid patches comprise DNA, RNA, combinationsthereof. In some embodiments, the nucleic acid patches comprise DNA. Insome embodiments, the nucleic acid patches comprise double-stranded DNA.In some embodiments, the nucleic acid patches comprise single-strandedDNA. In some embodiments, the nucleic acid patches comprise RNA. In someembodiments, the nucleic acid patches comprise single-stranded RNA. Insome embodiments, the nucleic acid patches comprise double-stranded RNA.

The nucleic acid patches may be any size. In some embodiments, thenucleic acid patches are up to about 1 μm² in size, up to about 10 μm²in size, up to about 100 μm² in size, up to about 1000 μm² in size, orup to about 10000 μm² in size. In some embodiments, the nucleic acidpatches are at least about 1 μm² in size, at least about 10 μm² in size,at least about 100 μm² in size, at least about 1000 μm² in size, or atleast about 10000 μm² in size. In some embodiments, the nucleic acidpatches are about 1 μm² in size, about 10 μm² in size, about 100 μm² insize, about 1000 μm² in size, or about 10000 μm² in size.

The nucleic acid patches may be separated by a defined distance. In someembodiments, the nucleic acid patches are separated by up to about 1 μm,up to about 10 μm, up to about 100 μm, up to about 1000 μm, or up toabout 10000 μm. In some embodiments, the nucleic acid patches areseparated by at least about 1 μm, at least about 10 μm, at least about100 μm, at least about 1000 μm, or at least about 10000 μm. In someembodiments, the nucleic acid patches are separated by about 1 μm, about10 μm, about 100 μm, about 1000 μm, or about 10000 μm.

The nucleic acid patches may be arranged on the surface in anyconfiguration. In some embodiments, the nucleic acid patches arearranged in a grid pattern. In some embodiments, the nucleic acidpatches are distributed randomly. In some embodiments, the nucleic acidpatches are arranged in a circular configuration.

The nucleic acid patches may be of any density on the surface. In someembodiments, the density of nucleic acid patches is at least 100patches/cm², at least 1000 patches/cm², at least 10000 patches/cm², atleast 100000 patches/cm², at least 1000000 patches/cm², or at least10000000 patches/cm². In some embodiments, the density of nucleic acidpatches is about 100 patches/cm², about 1000 patches/cm², about 10000patches/cm², about 100000 patches/cm², about 1000000 patches/cm², orabout 10000000 patches/cm².

The array device may be any size. In some embodiments, the surface areaof the device is at least 1 cm², at least 5 cm², at least 10 cm², atleast 25 cm², at least 50 cm², at least 100 cm², at least 500 cm², or atleast 1000 cm². In some embodiments, the surface area of the device isabout 1 cm², about 5 cm², about 10 cm², about 25 cm², about 50 cm²,about 100 cm², about 500 cm², or about 1000 cm². In some embodiments,the surface area of the device is at most 1 cm², at most 5 cm², at most10 cm², at most 25 cm², at most 50 cm², at most 100 cm², at most 500cm², or at most 1000 cm².

In one aspect, provided herein, is a method of performing a screen usingthe arrays described herein. In some embodiments, the method comprisesbinding nucleic acid encoded beads to the nucleic acid patches of thearray. In some embodiments, the method comprises sequencing the nucleicacid encoded beads. In some embodiments, cells are bound to the nucleicacid patches. In some embodiments, an assay is performed on the array.

The beads may contain an effector. In some embodiments the beadscomprise encoded effectors. In some embodiments, the beads comprisenucleic acid encoded effectors. In some embodiments, the effectors arereleased from the beads. In some embodiments, the effectors are releasedby cleaving a cleavable linker. In some embodiments, the cleavablelinker is cleaved by electromagnetic radiation. In some embodiments, thecleavable linker is cleaved by a cleaving reagent. In some embodiments,the method comprises adding a cleaving reagent to the nucleic acidpatches.

In some embodiments, reagents are added through the channels beneath thesurface. In some embodiments, the cleaving reagent is added through thechannels. In some embodiments, detection reagents are added through thechannels.

Sequencing the beads allows the locations of encoded beads in space tobe determined. In some embodiments, sequencing the beads allowsdetermination of the physical location of specific nucleic acid encodedbeads.

Any assay may be performed on the array. In some embodiments, the assayproduces a detectable signal. In some embodiments, the detectable signalis electromagnetic radiation. In some embodiments, the signal isfluorescence or luminescence.

The nucleic acid patches can bind any amount of cells or beads. In someembodiments, each nucleic acid patch binds a single bead. In someembodiments, each nucleic acid patch binds a single cell. In someembodiments, each nucleic acid patch binds a single bead and a singlecell. In some embodiments, each nucleic acid patch binds a plurality ofbeads. In some embodiments, each nucleic acid patch binds a plurality ofcells.

Numbered Embodiments

The following embodiments recite nonlimiting permutations ofcombinations of features disclosed herein. Other permutations ofcombinations of features are also contemplated. In particular, each ofthese numbered embodiments is contemplated as depending from or relatingto every previous or subsequent numbered embodiment, independent oftheir order as listed.

Embodiment 1: A method for screening an encoded effector, the methodcomprising: a) providing a sample, an encoded effector, and an encodingin an encapsulation; wherein the encoded effector is bound to a scaffoldby a cleavable linker; b) activating the cleavable linker using anactivating reagent; c) cleaving the cleavable linker so as to release apredetermined amount of the encoded effector; d) detecting a signal fromthe encapsulation, wherein the signal results from an interaction of theencoded effector and the sample; and e) sorting the encapsulation basedon the detection of the signal. Embodiment 2: The method of Embodiment1, wherein the activating reagent is provided with the encapsulation instep (a). Embodiment 3: The method of Embodiment 1, wherein theactivating reagent is added into the encapsulation. Embodiment 4: Themethod of Embodiment 3, wherein the activating reagent is added into theencapsulation by pico-injection. Embodiment 5: The method of Embodiment1, wherein the activating reagent is added to the encapsulation bydroplet merging, wherein the encapsulation is a droplet. Embodiment 6:The method of Embodiment 1, wherein the activating reagent is adisulfide reducing reagent. Embodiment 7: The method of Embodiment 1,wherein the activating reagent is a tetrazine. Embodiment 8: The methodof Embodiment 1, wherein the concentration of the activating reagentused to activate the cleavable linker is at most 100 picomolar (pM), atmost 500 pM, at most 1 nanomolar (nM), at most 10 nM, at most 100 nM, atmost 1 micromolar (04), at most 10 μM, at most 100 μM, at most 1millimolar (mM), at most 10 mM, at most 100 mM, or at most 500 mM.Embodiment 9: The method of Embodiment 1, wherein the activate reagentis added from a stock solution at least 2×, 5×, 10×, 20×, 30×, 50×,100×, 500×, or 1000× more concentrated than the desired finalconcentration in the encapsulation. Embodiment 10: The method ofEmbodiment 1, wherein the predetermined amount of effector released fromthe scaffold is to a concentration of at least 100 pM, at least 500 pM,at least 1 nM, at least 10 nM, at least 100 nM, at least 1 μM, at least10 μM, at least 100 μM, at least 1 mM. at least 10 mM, at least 50 mM,at least 100 mM, or at least 250 mM. Embodiment 11: The method ofEmbodiment 1, wherein the cleavable linker is a disulfide or substitutedtrans-cyclooctene. Embodiment 12: The method of Embodiment 1, whereinthe sample comprises at least one cell, a protein, an enzyme, a nucleicacid, a cellular lysate, a tissue extract, or combinations thereof.Embodiment 13: The method of Embodiment 12, wherein the sample is one ormore cells, a protein, or an enzyme. Embodiment 14: The method ofEmbodiment 1, wherein the scaffold is a bead, a fiber, a nanofibrousscaffold, a molecular cage, a dendrimer, or a multi-valent molecularassembly. Embodiment 15: The method of Embodiment 14, wherein thescaffold is polymer-bead, a glass bead, a metal bead, or a magneticbead. Embodiment 16: The method of Embodiment 15, wherein the bead isabout 1 μm to about 100 μm in diameter. Embodiment 17: The method ofEmbodiment 15, wherein the bead is about 1 μm to about 20 μm indiameter. Embodiment 18: The method of Embodiment 1, wherein the encodedeffector is a peptide, a compound, protein, an enzyme, a macrocyclecompound, or a nucleic acid. Embodiment 19: The method of Embodiment 18,wherein the encoded effector is a non-natural peptide. Embodiment 20:The method of Embodiment 18, wherein the encoded effector is a polymer.Embodiment 21: The method of Embodiment 18, wherein the compound is adrug-like small molecule. Embodiment 22: The method of Embodiment 1,wherein the encapsulation is a droplet. Embodiment 23: The method ofEmbodiment 22, wherein the droplet is at most 1 picoliter, at most 10picoliters, at most 100 picoliters, at most 1 nanoliter, at most 10nanoliters, at most 100 nanoliters, or at most 1 microliter in volume.Embodiment 24: The method of Embodiment 1, wherein the signal compriseselectromagnetic radiation, thermal radiation, a visual change in thesample, or combinations thereof. Embodiment 25: The method of Embodiment24, wherein the electromagnetic radiation is in the visible spectrum.Embodiment 26: The method of Embodiment 24, wherein the electromagneticradiation is fluorescence or luminescence. Embodiment 27: The method ofEmbodiment 26, wherein the signal is fluorescence emitted by a TaqManprobe or a molecular beacon. Embodiment 28: The method of Embodiment 24,wherein the signal comprises thermal radiation detected with an infraredcamera. Embodiment 29: The method of Embodiment 24, wherein the signalcomprises a morphological or visual change in the sample measured byrecording a series of images of the encapsulation. Embodiment 30: Themethod of Embodiment 1, further comprising incubating the encapsulationfor a period of time to allow the effector and the sample to interact.Embodiment 31: The method of Embodiment 30, wherein the period of timeis controlled by a residence time as the encapsulation travels through amicrofluidic channel, wherein the residence time of each encapsulationis within a maximum dispersion ratio of the incubation period of timefor the plurality of encapsulations, wherein the dispersion ratio isbased on a deviation about an average residence time of the plurality ofencapsulations passing through a region of the microfluidic device.Embodiment 32: The method of Embodiment 31, wherein the maximumdispersion is at most from about 3% to about 10%. Embodiment 33: Themethod of Embodiment 1, wherein sorting the encapsulation comprisesplacing the droplet into a first collection tube if the signal is at orabove a predetermined threshold or placing the droplet into a secondcollection tube if the signal is below a predetermined threshold.Embodiment 34: The method of Embodiment 1, wherein sorting theencapsulation comprises using a waveform pulse generator to move theencapsulation to a collection tube by an electrical field gradient, bysound, by a diaphragm, by modifying geometry of the microfluidicchannel, or by changing the pressure of the microfluidic channel.Embodiment 35: The method of Embodiment 1, wherein the encapsulation isan emulsion in an oil. Embodiment 36: The method of Embodiment 1,wherein the encoding is a nucleic acid and the method further comprisesthe step of sequencing the encoding nucleic acid. Embodiment 37: Themethod of Embodiment 36, wherein the encoding is cleaved from thescaffold prior to sequencing. Embodiment 38: The method of Embodiment37, wherein cleaving the nucleic acid encoding from the scaffoldcomprises cleaving a cleavable linker with a cleaving reagent or throughelectromagnetic radiation.

Embodiment 39: A method for screening an encoded effector, the methodcomprising: a) providing a sample, an encoded effector, and an encodingin an encapsulation; wherein the encoded effector is bound to a scaffoldby a cleavable linker; b) cleaving the cleavable linker with a cleavingreagent, wherein the cleaving reagent is added at a concentrationconfigured to release a predetermined amount of the encoded effector; c)detecting a signal from the encapsulation, wherein the signal resultsfrom an interaction of the encoded effector and the sample; and d)sorting the encapsulation based on the detection of the signal.Embodiment 40: The method of Embodiment 39, wherein the cleaving reagentis added to the encapsulation by pico-injection. Embodiment 41: Themethod of Embodiment 39, wherein the cleaving reagent is added to theencapsulation at a step separate from forming the encapsulation.Embodiment 42: The method of Embodiment 39, wherein the cleaving reagentis added to the encapsulation using a solution comprising the cleavingreagent and the sample prior to formation of the encapsulation.Embodiment 43: The method of Embodiment 39, wherein the concentration ofcleaving reagent used to cleave the cleavable linker is at most 100picomolar (pM), at most 500 pM, at most 1 nanomolar (nM), at most 10 nM,at most 100 nM, at most 1 micromolar (μM), at most 10 μM, at most 100μM, at most 1 millimolar (mM), at most 10 mM, at most 100 mM, or at most500 mM. Embodiment 44: The method of Embodiment 39, wherein the cleavingreagent is added from a stock solution at least 2×, 5×, 10×, 20×, 30×,50×, 100×, 500×, or 1000× more concentrated than the desired finalconcentration in the encapsulation. Embodiment 45: The method ofEmbodiment 39, wherein the predetermined amount of effector releasedfrom the scaffold is to a concentration of at least 100 pM, at least 500pM, at least 1 nM, at least 10 nM, at least 100 nM, at least 1 μM, atleast 10 μM, at least 100 μM, at least 1 mM. at least 10 mM, at least 50mM, at least 100 mM, or at least 250 mM. Embodiment 46: The method ofEmbodiment 39, wherein the cleavable linker is a disulfide orsubstituted trans-cyclooctene. Embodiment 47: The method of Embodiment39, wherein the cleaving reagent is a disulfide reducing reagent.Embodiment 48: The method of Embodiment 39, wherein the cleaving reagentis a tetrazine. Embodiment 49: The method of Embodiment 39, wherein thesample comprises at least one cell, a protein, an enzyme, a nucleicacid, a cellular lysate, a tissue extract, or combinations thereof.Embodiment 50: The method of Embodiment 49, wherein the sample is one ormore cells, a protein, or an enzyme. Embodiment 51: The method ofEmbodiment 49, wherein the scaffold is a bead, a fiber, a nanofibrousscaffold, a molecular cage, a dendrimer, or a multi-valent molecularassembly. Embodiment 52: The method of Embodiment 51, wherein thescaffold is polymer-bead, a glass bead, a metal bead, or a magneticbead. Embodiment 53: The method of Embodiment 52, wherein the bead isabout 1 μm to about 100 μm in diameter. Embodiment 54: The method ofEmbodiment 52, wherein the bead is about 1 μm to about 20 μm indiameter. Embodiment 55: The method of Embodiment 39, wherein theencoded effector is a peptide, a compound, protein, an enzyme, amacrocycle compound, or a nucleic acid. Embodiment 56: The method ofEmbodiment 55, wherein the encoded effector is a non-natural peptide.Embodiment 57: The method of Embodiment 55, wherein the encoded effectoris a polymer. Embodiment 58: The method of Embodiment 55, wherein thecompound is a drug-like small molecule. Embodiment 59: The method ofEmbodiment 39, wherein the encapsulation is a droplet. Embodiment 60:The method of Embodiment 59, wherein the droplet is at most 1 picoliter,at most 10 picoliters, at most 100 picoliters, at most 1 nanoliter, atmost 10 nanoliters, at most 100 nanoliters, or at most 1 microliter involume. Embodiment 61: The method of Embodiment 39, wherein the signalcomprises electromagnetic radiation, thermal radiation, a visual changein the sample, or combinations thereof. Embodiment 62: The method ofEmbodiment 61, wherein the electromagnetic radiation is in the visiblespectrum. Embodiment 63: The method of Embodiment 61, wherein theelectromagnetic radiation is fluorescence or luminescence. Embodiment64: The method of Embodiment 63, wherein the signal is fluorescenceemitted by a TaqMan probe or a molecular beacon. Embodiment 65: Themethod of Embodiment 61, wherein the signal comprises thermal radiationdetected with an infrared camera. Embodiment 66: The method ofEmbodiment 61, wherein the signal comprises a morphological or visualchange in the sample measured by recording a series of images of theencapsulation. Embodiment 67: The method of Embodiment 39, furthercomprising incubating the encapsulation for a period of time to allowthe effector and the sample to interact. Embodiment 68: The method ofEmbodiment 67, wherein the period of time is controlled by a residencetime as the encapsulation travels through a microfluidic channel,wherein the residence time of each encapsulation is within a maximumdispersion ratio of the incubation period of time for the plurality ofencapsulations, wherein the dispersion ratio is based on a deviationabout an average residence time of the plurality of encapsulationspassing through a region of the microfluidic device. Embodiment 69: Themethod of Embodiment 68, wherein the maximum dispersion is at most fromabout 3% to about 10%. Embodiment 70: The method of Embodiment 39,wherein sorting the encapsulation comprises placing the droplet into afirst collection tube if the signal is at or above a predeterminedthreshold or placing the droplet into a second collection tube if thesignal is below a predetermined threshold. Embodiment 71: The method ofEmbodiment 39, wherein sorting the encapsulation comprises using awaveform pulse generator to move the encapsulation to a collection tubeby an electrical field gradient, by sound, by a diaphragm, by modifyinggeometry of the microfluidic channel, or by changing the pressure of themicrofluidic channel. Embodiment 72: The method of Embodiment 39,wherein the encapsulation is an emulsion in an oil. Embodiment 73: Themethod of Embodiment 39, wherein the encoding is a nucleic acid and themethod further comprises the step of sequencing the encoding nucleicacid. Embodiment 74: The method of Embodiment 73, wherein the encodingis cleaved from the scaffold prior to sequencing. Embodiment 75: Themethod of Embodiment 74, wherein cleaving the nucleic acid encoding fromthe scaffold comprises cleaving a cleavable linker with a cleavingreagent or through electromagnetic radiation.

Embodiment 76: A method for screening an encoded effector, the methodcomprising: a) providing at least one cell and a scaffold in anencapsulation, wherein the scaffold comprises an encoded effector boundto the scaffold by a photocleavable linker and a nucleic acid encodingthe effector; b) cleaving the photocleavable linker to release theencoded effector from the scaffold; and c) detecting a signal from thedroplet, wherein the signal results from an interaction between theencoded effector and the at least one cell. Embodiment 77: The method ofEmbodiment 76, further comprising sorting the encapsulation based on thedetection of the signal. Embodiment 78: The method of Embodiment 77,wherein sorting the droplet comprises using a waveform pulse generatorto move the droplet to a collection tube by an electrical fieldgradient, by sound, by a diaphragm, by modifying geometry of themicrofluidic channel, or by changing the pressure of the microfluidicchannel. Embodiment 79: The method of Embodiment 77, further comprisingidentifying the encoded effector by sequencing the nucleic acid encodingthe effector. Embodiment 80: The method of Embodiment 76, furthercomprising barcoding the nucleic acid encoding the effector. Embodiment81: The method of Embodiment 80, wherein the barcoding is via theaddition of a reagent into the droplet. Embodiment 82: The method ofEmbodiment 76, wherein cleaving the photocleavable linker releases apre-determined amount of the encoded effector into the droplet.Embodiment 83: The method of Embodiment 76, wherein the photocleavablelinker is cleaved using electromagnetic radiation. Embodiment 84: Themethod of Embodiment 76, wherein cleaving the photocleavable linkercomprises exposing the encapsulation to a light from a light source.Embodiment 85: The method of Embodiment 84, wherein the light is acalibrated amount of light. Embodiment 86: The method of Embodiment 84,wherein the light is UV light. Embodiment 87: The method of Embodiment84, wherein the light intensity of a light is from about 0.01 J/cm² toabout 200 J/cm². Embodiment 88: The method of Embodiment 76, whereindetecting the signal comprises detecting morphological changes in thesample measured by recording a series of images of the droplet ordetecting fluorescence emitted by a molecular beacon or probe.Embodiment 89: The method of Embodiment 76, wherein the interactionbetween the encoded effector and the cell comprises inhibition of one ormore cellular components. Embodiment 90: The method of Embodiment 76,further comprising identifying the encoded effector by sequencing thenucleic acid encoding the effector. Embodiment 91: The method ofEmbodiment 76, wherein two or more cells are provided with the scaffold.Embodiment 92: The method of Embodiment 76, further comprising providingan activating reagent to activate the photocleavable linker, so as toenable the photocleavable linker to be cleaved from the encodedeffector. Embodiment 93: The method of Embodiment 92, wherein theactivating reagent is provided with the encapsulation. Embodiment 94:The method of Embodiment 92, wherein the activating reagent is addedinto the encapsulation through pico injection or droplet merging.Embodiment 95: The method Embodiment 76, further comprising the step oflysing the one or more cells. Embodiment 96: The method of Embodiment95, wherein lysing the one or more cells comprises adding lysis bufferto the encapsulation. Embodiment 97: The method of Embodiment 96,wherein the lysis buffer is added to the encapsulation bypico-injection. Embodiment 98: The method of Embodiment 76, wherein thescaffold is a bead, a fiber, a nanofibrous scaffold, a molecular cage, adendrimer, or a multi-valent molecular assembly. Embodiment 99: Themethod of Embodiment 98, wherein the scaffold is polymer-bead, a glassbead, a metal bead, or a magnetic bead. Embodiment 100: The method ofEmbodiment 98, wherein the bead is about 1 μm to about 100 μm indiameter. Embodiment 101: The method of Embodiment 98, wherein the beadis about 1 μm to about 20 μm in diameter. Embodiment 102: The method ofEmbodiment 76, wherein the encoded effector is a peptide, a compound,protein, an enzyme, a macrocycle compound, or a nucleic acid. Embodiment103: The method of Embodiment 102, wherein the encoded effector is anon-natural peptide. Embodiment 104: The method of Embodiment 102,wherein the encoded effector is a polymer. Embodiment 105: The method ofEmbodiment 102, wherein the compound is a drug-like small molecule.Embodiment 106: The method of Embodiment 76, wherein the encapsulationis a droplet. Embodiment 107: The method of Embodiment 106, wherein thedroplet is at most 1 picoliter, at most 10 picoliters, at most 100picoliters, at most 1 nanoliter, at most 10 nanoliters, at most 100nanoliters, or at most 1 microliter in volume. Embodiment 108: Themethod of Embodiment 76, further comprising incubating the droplet for aperiod of time to allow the effector and the at least one cell tointeract. Embodiment 109: The method of Embodiment 108, wherein theperiod of time is controlled by a residence time as the encapsulationtravels through a microfluidic channel, wherein the residence time ofeach encapsulation is within a maximum dispersion ratio of theincubation period of time for the plurality of encapsulations, whereinthe dispersion ratio is based on a deviation about an average residencetime of the plurality of encapsulations passing through a region of themicrofluidic device. Embodiment 110: The method of Embodiment 109,wherein the maximum dispersion is at most from about 3% to about 10%.The method of Embodiment 33, wherein sorting the droplet comprisesplacing the droplet into a first collection tube if the signal is at orabove a predetermined threshold or placing the droplet into a secondcollection tube if the signal is below a predetermined threshold.Embodiment 111: The method of Embodiment 76, wherein the signalcomprises electromagnetic radiation, thermal radiation, a visual changein the sample, or combinations thereof. Embodiment 112: The method ofEmbodiment 111, wherein the electromagnetic radiation is in the visiblespectrum. Embodiment 113: The method of Embodiment 111, wherein theelectromagnetic radiation is fluorescence or luminescence. Embodiment114: The method of Embodiment 113, wherein the signal is fluorescenceemitted by a TaqMan probe or a molecular beacon. Embodiment 115: Themethod of Embodiment 111, wherein the signal comprises thermal radiationdetected with an infrared camera. Embodiment 116: The method ofEmbodiment 111, wherein the signal comprises a morphological or visualchange in the sample measured by recording a series of images of theencapsulation.

Embodiment 117: A method for screening an encoded effector, the methodcomprising: a) providing a sample, an encoded effector, and an encodingin an encapsulation; b) detecting a signal resulting from an interactionbetween the effector and sample, wherein detecting the signalcomprises 1) detecting morphological changes in the sample measured byrecording a series of images of the encapsulation, 2) detectingfluorescence emitted by a molecular beacon or probe, or 3) combinationsthereof, and c) sorting the encapsulation based on the detection of thesignal. Embodiment 118: The method of Embodiment 117, wherein the signalcomprises detecting a morphological or visual change in the samplemeasured by recording a series of images of the encapsulation.Embodiment 119: The method of Embodiment 118, wherein the encapsulationfurther comprises a detection reagent. Embodiment 120: The method ofEmbodiment 119, wherein the detection reagent comprises an intercalationdye. Embodiment 121: The method of Embodiment 120, wherein theintercalation dye comprises ethidium bromide, propidium iodide, crystalviolet, a dUTP-conjugated probe, DAPI (4′,6-diamidino-2-phenylindole),7-AAD (7-aminoactinomycin D), Hoechst 33258, Hoechst 33342, Hoechst34580, combinations thereof, or derivatives thereof. Embodiment 122: Themethod of Embodiment 118, further comprising superimposing the series ofimages of the sample in the encapsulation. Embodiment 123: The method ofEmbodiment 117, wherein the signal comprises detecting fluorescenceemitted by a molecular beacon or TaqMan probe. Embodiment 124: Themethod of Embodiment 123, wherein the signal comprises detectingfluorescence emitted by a molecular beacon, wherein molecular beacon iscomplementary to a portion of a target nucleic acid sequence of thesample. Embodiment 125: The method of Embodiment 123, wherein the signalcomprises detecting fluorescence emitted by a TaqMan probe, wherein theTaqMan probe is complementary to a portion of a target nucleic acidsequence. Embodiment 126: The method of Embodiment 123, wherein theencapsulation further comprises a Taq polymerase. Embodiment 127: Themethod of Embodiment 123, wherein the TaqMan probe or molecular beaconis added to the encapsulation by pico-injection. Embodiment 128: Themethod of Embodiment 117, wherein the encoded effector is attached to ascaffold. Embodiment 129: The method of Embodiment 128, wherein thescaffold is a bead, a fiber, a nanofibrous scaffold, a molecular cage, adendrimer, or a multi-valent molecular assembly. Embodiment 130: Themethod of Embodiment 129, wherein the scaffold is polymer-bead, a glassbead, a metal bead, or a magnetic bead. Embodiment 131: The method ofEmbodiment 128, wherein the encoded effector is covalently bound to thescaffold. Embodiment 132: The method of Embodiment 128, wherein theencoded effector is bound to the scaffold by a cleavable linker.Embodiment 133: The method of Embodiment 132, wherein the cleavablelinker is a disulfide or substituted trans-cyclooctene. Embodiment 134:The method of Embodiment 132, further comprising cleaving the cleavablelinker. Embodiment 135: The method of Embodiment 134, wherein the numberof encoded effectors cleaved from the scaffold is controlled by theintensity or duration of exposure to electromagnetic radiation.Embodiment 136: The method of Embodiment 134, wherein the number ofencoded effectors cleaved from the scaffold is controlled by controllingthe concentration of a cleaving reagent in the encapsulation. Embodiment137: The method of Embodiment 136, wherein the cleaving reagent is addedby pico-injection. Embodiment 138: The method of Embodiment 134, whereinthe cleavable linker is activated through interaction with an activatingreagent, thereby enabling the cleavable linker to be cleaved. Embodiment139: The method of Embodiment 134, wherein the encoded effectors arereleased to a desired concentration within the encapsulation. Embodiment140: The method of Embodiment 117, further comprising incubating theencapsulation for a period of time to allow the encoded effector and thesample to interact. Embodiment 141: The method of Embodiment 140,wherein the period of time is at least 1 minute, at least 10 minutes, atleast 1 hour, at least 4 hours, or at least 1 day. Embodiment 142: Themethod of Embodiment 140, wherein the period of time is controlled by aresidence time as the encapsulation travels through a microfluidicchannel, wherein the residence time of each encapsulation is within amaximum dispersion ratio of the incubation period of time for theplurality of encapsulations, wherein the dispersion ratio is based on adeviation about an average residence time of the plurality ofencapsulations passing through a region of the microfluidic device.Embodiment 143: The method of Embodiment 142, wherein the maximumdispersion is at most from about 3% to about 10%. Embodiment 144: Themethod of Embodiment 142, wherein the residence time is controlled by aflow rate through the microfluidic channel, a geometry of themicrofluidic channel, a valve in the microfluidic channel, or byremoving the one or more droplets from the microfluidic channel andtransferring the one or more droplets to a separate vessel. Embodiment145: The method of Embodiment 117, wherein the encoded effectorcomprises a compound, a peptide, a protein, an enzyme, a macrocyclecompound, or a nucleic acid. Embodiment 146: The method of Embodiment145, wherein the encoded effector is a non-natural peptide. Embodiment147: The method of Embodiment 145, wherein the encoded effector is apolymer. Embodiment 148: The method of Embodiment 145, wherein thecompound is a drug-like small molecule. Embodiment 149: The method ofEmbodiment 117, wherein the sample comprises one or more cells.Embodiment 150: The method of Embodiment 149, further comprising thestep of lysing the one or more cells. Embodiment 151: The method ofEmbodiment 117, wherein detecting the signal comprises providing one ormore droplets through a microfluidic channel comprising a detector.Embodiment 152: The method of Embodiment 117, wherein sorting theencapsulation comprises placing the encapsulation into a firstcollection tube if the signal is at or above a predetermined thresholdor placing the encapsulation into a second collection tube if the signalis below a predetermined threshold. Embodiment 153: The method ofEmbodiment 117, wherein sorting the encapsulation comprises using awaveform pulse generator to move encapsulation to a collection tube byan electrical field gradient, by sound, by a diaphragm, by modifyinggeometry of a microfluidic channel, or by changing the pressure of themicrofluidic channel. Embodiment 154: The method of Embodiment 117,wherein the encoding comprises a nucleic acid. Embodiment 155: Themethod of Embodiment 154, further comprising sequencing the encodingnucleic acid. Embodiment 156: The method of Embodiment 155, wherein theencoding is cleaved from the scaffold prior to sequencing. Embodiment157: The method of Embodiment 156, wherein cleaving the nucleic acidencoding from the scaffold comprises cleaving a cleavable linker with acleaving reagent or through electromagnetic radiation. Embodiment 158:The method of Embodiment 117, wherein the encapsulation is a droplet.Embodiment 159: The method of Embodiment 158, wherein the droplet the isat most 1 picoliter, at most 10 picoliters, at most 100 picoliters, atmost 1 nanoliter, at most 10 nanoliters, at most 100 nanoliters, or atmost 1 microliter in volume. Embodiment 160: The method of Embodiment117, wherein the encapsulation is an emulsion in an oil. Embodiment 161:The method of Embodiment 160, wherein the oil is a silicone oil,fluorosilicone oil, hydrocarbon oil, mineral oil, paraffin oil,halogenated oil, or any combination thereof. Embodiment 162: The methodof Embodiment 117, further comprising injecting one or more reagentsinto one or more encapsulations. Embodiment 163: The method ofEmbodiment 162, wherein the one or more reagents are injected bypico-injection or droplet merging. Embodiment 164: The method ofEmbodiment 162, wherein injecting the one or more reagents furthercomprises monitoring the one or more encapsulations in flow, wherein theone or more reagents are injected at the same frequency at which the oneor more encapsulations are passing an injection site. Embodiment 165:The method of Embodiment 140, wherein a rate of injection of the one ormore reagents is determined by a flow rate of the one or moreencapsulations.

Embodiment 166: A method for detecting one or more cellular nucleic acidusing a nucleic acid encoded effector screen, the method comprising: a)providing an encoded effector, a nucleic acid encoding the encodedeffector, and one or more cells comprising the one or more cellularnucleic acids, wherein the encoded effector, nucleic acid encoding andthe one or more cells are provided in an encapsulation; b) incubatingthe encapsulation for a period of time to allow for the encoded effectorand the one or more cells to interact, thereby producing a signal; c)transferring at least one cellular nucleic acid of the one or morecellular nucleic acids to the nucleic acid encoding; d) detecting thesignal; and e) sorting the encapsulation based on the detection of thesignal.

Embodiment 167: The method Embodiment 166, further comprising the stepof lysing the one or more cells. Embodiment 168: The method ofEmbodiment 167, wherein lysing the one or more cells comprises addinglysis buffer to the encapsulation. Embodiment 169: The method ofEmbodiment 168, wherein the lysis buffer is added to the encapsulationby pico-injection. Embodiment 170: The method of Embodiment 166, whereinthe one or more cellular nucleic acids comprise DNA, RNA, orcombinations thereof. Embodiment 171: The method of Embodiment 166,wherein the one or more cellular nucleic acids comprise mRNA. Embodiment172: The method of Embodiment 664, wherein the one or more cellularnucleic acids are added to the nucleic acid encoding as antibody-DNAconstructs, proximity ligation products, or proximity extensionproducts. Embodiment 173: The method of Embodiment 166, whereintransferring the at least one cellular nucleic acid to the nucleic acidencoding comprises annealing, ligating, amplifying, or chemicallycrosslinking the at least one cellular nucleic acid to the nucleic acidencoding. Embodiment 174: The method Embodiment 166, whereintransferring the at least one cellular nucleic acid to the nucleic acidencoding allows for quantification of the amount of the one or morecellular nucleic acids encapsulated with the nucleic acid encodedeffector. Embodiment 175: The method of Embodiment 166, wherein aplurality of different cellular nucleic acids are transferred to thenucleic acid encoding. Embodiment 176: The method of Embodiment 166,further comprising adding one or more reagents for transferring the atleast one cellular nucleic acid to the nucleic acid encoding. Embodiment177: The method of Embodiment 176, wherein the one or more reagents areprovided in the encapsulation in step (a). Embodiment 178: The method ofEmbodiment 176, wherein the one or more reagents are added during theincubation step or after the incubation step. Embodiment 179: The methodof Embodiment 176, wherein the one or more reagents are added by dropletmerging, pico-injection, or interaction with solid-phase particlescomprising the one or more reagents. Embodiment 180: The method ofEmbodiment 176, wherein the one or more reagents comprises an enzyme.Embodiment 181: The method of Embodiment 180, wherein the enzyme is aligase, a polymerase, a restriction enzyme, or a recombinase. Embodiment182: The method of Embodiment 176, wherein the one or more reagentscomprises assay detection reagents, labelling reagents, antibodies,target effectors, cell lysis reagents, nucleic acid ligation reagents,amplification reagents, or combinations thereof. Embodiment 183: Themethod of Embodiment 176, wherein the one or more reagents are onlyadded if a signal is detected. Embodiment 184: The method of Embodiment166, wherein the signal is electromagnetic radiation, thermal radiation,or a visual change in the sample. Embodiment 185: The method ofEmbodiment 166, wherein detecting the signal comprises providing theencapsulation through a microfluidic channel equipped with a detector.Embodiment 186: The method of Embodiment 166, wherein sorting theencapsulation is based on the level, presence, or absence of the signal.Embodiment 187: The method of Embodiment 166, wherein the period of timeis controlled by a residence time as the encapsulation travels through amicrofluidic channel, wherein the residence time of each encapsulationis within a maximum dispersion ratio of the incubation period of timefor the plurality of encapsulations, wherein the dispersion ratio isbased on a deviation about an average residence time of the plurality ofencapsulations passing through a region of the microfluidic device.Embodiment 188: The method of Embodiment 187, wherein the maximumdispersion is at most from about 3% to about 10%. Embodiment 189: Themethod of Embodiment 166, wherein the period of time is at least 1minute, at least 10 minutes, at least 1 hour, at least 4 hours, or atleast 1 day. Embodiment 190: The method of Embodiment 166, wherein theencapsulation is a droplet, an emulsion, a picowell, a microwell, abubble, or a microfluidic confinement. Embodiment 191: The method ofEmbodiment 166, wherein the encapsulation is a droplet. Embodiment 192:The method of Embodiment 191, wherein the droplet is at most 1picoliter, at most 10 picoliters, at most 100 picoliters, at most 1nanoliter, at most 10 nanoliters, at most 100 nanoliters, or at most 1microliter in volume. Embodiment 193: The method of Embodiment 191,wherein the droplet is suspended in an emulsion. Embodiment 194: Themethod of Embodiment 1166, wherein the effector comprises a compound, apeptide, a protein, an enzyme, a macrocycle compound, or a nucleic acid.Embodiment 195: The method of Embodiment 166, further comprising 1)amplifying the nucleic acid encoding the encoded effector with thetransferred at least one cellular nucleic acid, 2) sequencing thenucleic acid encoding with the transferred at least one cellular nucleicacid, 3) quantifying the at least one cellular nucleic acid, or anycombination thereof. Embodiment 196: The method of Embodiment 166,wherein the encoded effector is attached to a scaffold. Embodiment 197:The method of Embodiment 196, wherein the scaffold is a bead. Embodiment198: The method of Embodiment 196, wherein the scaffold is apolymer-bead, a glass bead, a metal bead, a molecular cage, or amulti-valent molecular assembly. Embodiment 199: The method ofEmbodiment 196, wherein the encoded effector is attached to the scaffoldby a cleavable linker. Embodiment 200: The method of Embodiment 199,wherein the cleavable linker is a photocleavable linker. Embodiment 201:The method of Embodiment 199, wherein the encoded effector is covalentlyattached to the cleavable linker. Embodiment 201: The method ofEmbodiment 199, further comprising cleaving the cleavable linker.Embodiment 202: The method of Embodiment 199, wherein the nucleic acidencoding is attached to the scaffold by a second cleavable linker.Embodiment 203: The method of Embodiment 203, further comprisingcleaving the second cleavable linker.

Embodiment 205: A method for screening a nucleic acid encoded protein,the method comprising: a) providing an encapsulation comprising: i) anucleic acid encoding attached to a scaffold, the nucleic acid encodingcomprises an encoding barcode and a coding section for the expression ofan encoded effector protein, and ii) an expression system for theproduction of the encoded effector protein; b) expressing the encodedeffector protein within the encapsulation; c) detecting the signalproduced from an interaction with the encoded effector protein and oneor more detection reagents disposed within the encapsulation; and d)sorting the encapsulation based on the signal. Embodiment 206: Themethod of Embodiment 205, further comprising the step of sequencing thenucleic acid encoding based on the sorted encapsulation. Embodiment 207:The method of Embodiment 205, wherein the encoded effector protein is asignaling protein, an enzyme, a binding protein, an antibody or antibodyfragment, a structural protein, a storage protein, or a transportprotein, or any mutant thereof. Embodiment 208: The method of Embodiment205, wherein the encoded effector protein is an enzyme or enzyme mutantbeing screened for an enzymatic activity. Embodiment 209: The method ofEmbodiment 208, wherein the enzymatic activity comprises oxidation,reduction, ligation, polymerization, bond cleavage, bond formation, orisomerization. Embodiment 210: The method of Embodiment 205, wherein theencoded effector protein is an amino acid dehydrogenase, a natural aminedehydrogenase, an opine dehydrogenase, or an imine reductase. Embodiment211: The method of Embodiment 205, wherein the interaction between theencoded effector protein and the one or more detection reagentscomprises forming a bond between 1) a first molecular probe from a firstdetection reagent and second molecular probe from a second detectionreagent of the one or more reagents, or 2) one or more chemicalcompounds for a first detection reagent and one or more chemicalcompounds from a second detection reagent. Embodiment 212: The method ofEmbodiment 211, wherein the bond is a covalent bond. Embodiment 213: Themethod of Embodiment 211, wherein the bond is an irreversible covalentbond. Embodiment 214: The method of Embodiment 211, wherein the firstreagent and the second reagent exhibit a fluorescent signal when thefirst and second molecular probes are bound together. Embodiment 215:The method of Embodiment 214, wherein the fluorescent signal is due tofluorescence resonance energy transfer (FRET), bioluminescence resonanceenergy transfer (BRET), lanthanide chelate excite time resolvedfluorescence resonance energy transfer (LANCE TR-FRET), or an amplifiedluminescent proximity homogeneous assay. Embodiment 216: The method ofEmbodiment 211, wherein the first and second detection reagents comprisechemical compounds. Embodiment 217: The method of Embodiment 211,wherein the first and second reagents comprise a FRET pair or afluorophore/quencher pair. Embodiment 218: The method of Embodiment 217,wherein the first and second detection reagents comprise fluorophores orquenchers independently selected from 4-(4-dimethylaminophenyl azo),5-((3-aminoethyl)amino)-1-napthalene sulfonic acid,5-((2-aminoethyl)amino)-1-napthalene sulfonic acid (EDANS),4-(dimethylaminoazo)benzene-4-carboxylic acid (DABCYL), andfluorescein-isothiocyanate (FITC), or derivatives thereof. Embodiment219: The method of Embodiment 217, wherein the FRET pair orfluorophore/quencher pair comprise different fluorophores. Embodiment220: The method of Embodiment 217, wherein the FRET pairing is duplicatecopies of the same fluorophore. Embodiment 221: The method of Embodiment211, wherein forming of the bond comprising an imine reduction.Embodiment 222: The method of Embodiment 221, wherein the iminereduction is enantiospecific. Embodiment 223: The method of Embodiment211, wherein the encapsulation further comprises a reporter enzyme.Embodiment 224: The method of Embodiment 223, wherein the reporterenzyme reacts with another reagent to produce a functional readout.Embodiment 225: The method of Embodiment 223, wherein the bond betweenthe first and second molecular probes creates a new molecule thatinhibits the reporter enzyme. Embodiment 226: The method of Embodiment205, wherein the scaffold is a bead, a fiber, a nanofibrous scaffold, amolecular cage, a dendrimer, or a multi-valent molecular assembly.Embodiment 227: The method of Embodiment 226, wherein the scaffold is abead. Embodiment 228: The method of Embodiment 226, wherein the scaffoldis polymer-bead, a glass bead, a metal bead, or a magnetic bead.Embodiment 229: The method of Embodiment 205, wherein the encapsulationis a droplet, an emulsion, a picowell, a macrowell, a microwell, abubble, or a microfluidic confinement. Embodiment 230: The method of anyEmbodiment 229, wherein the encapsulation is a droplet. Embodiment 231:The method of Embodiment 230, wherein the droplet is at most 1picoliter, at most 10 picoliters, at most 100 picoliters, at most 1nanoliter, at most 10 nanoliters, at most 100 nanoliters, or at most 1microliter in volume. Embodiment 232: The method of Embodiment 205,wherein the expression system comprises an in vitrotranscription/translation system. Embodiment 233: The method ofEmbodiment 205, wherein the one or more detection reagents are addedthrough pico-injection or droplet merging. Embodiment 234: The method ofEmbodiment 205, further comprising incubating the encapsulation for aperiod of time after the one or more detection reagents have been added.Embodiment 235: The method of Embodiment 205, wherein detecting thesignal comprises providing the encapsulation through a microfluidicchannel equipped with a detector. Embodiment 236: A method of screeninga library of nucleic acid encoded proteins, the method comprisingperforming the screen of any of Embodiments 205-235 against a library ofnucleic acid encoded proteins, wherein the library of nucleic acidencoded proteins comprises a plurality of different mutant versions ofthe nucleic acid encoded protein. Embodiment 237: The method ofEmbodiment 236, wherein each mutant version of the nucleic acid encodedprotein is encoded by a unique barcode.

Embodiment 238: A method for normalizing the results of a nucleic acidencoded library screen comprising: a) providing a plurality of screenedencoded effectors and corresponding scaffolds in a plurality ofencapsulations, wherein each scaffold is bound to one or more nucleicacid encodings that encode a corresponding screened encoded effector; b)lysing the plurality of encapsulations; c) removing contents unbound tothe plurality of scaffolds; d) suspending the plurality of scaffolds ina liquid medium; e) encapsulating the plurality of scaffolds in aplurality of new encapsulations, wherein each new encapsulationencapsulates one or more scaffolds of the plurality of scaffolds; and f)amplifying the one or more nucleic acid encodings of each scaffold toform corresponding amplified nucleic acid encodings, such that theamplified nucleic encodings within the plurality of new encapsulationsare limited to the contained encoding scaffold(s) and the reagent(s)within the plurality of new encapsulations. Embodiment 239: The methodof Embodiment 238, wherein 90% of the plurality of new encapsulationshave a concentration of amplified nucleic acid encodings within 10% ofan average concentration of the amplified nucleic acid encodings in theplurality of new encapsulations. Embodiment 240: The method ofEmbodiment 238, wherein providing a plurality of screened encodedeffectors comprises performing a screen of a pre-screened nucleic acidencoded library. Embodiment 241: The method of Embodiment 240, whereinperforming the screen comprises a sorting step to separate nucleic acidencoded effectors from the pre-screened nucleic acid encoded librarythat displayed a positive result in the screen. Embodiment 242: Themethod of Embodiment 240, wherein the plurality of screened encodedeffectors comprises the nucleic acid encoded effectors that displayed apositive result in the screen of the pre-screened nucleic acid encodedlibrary. Embodiment 243: The method of Embodiment 238, wherein lysingthe plurality of encapsulations comprises introducing a demulsifyingreagent, filtration, centrifugation, or sonication to an emulsioncontaining the plurality of encapsulations. Embodiment 244: The methodof Embodiment 243, wherein the demulsifying reagent is an acid or asalt. Embodiment 245: The method of Embodiment 243, wherein thedemulsifying reagent is sulfuric acid or hydrochloric acid. Embodiment246: The method of Embodiment 243, wherein the demulsifying reagent issodium chloride, potassium pyrophosphate, or sodium sulfate. Embodiment247: The method of Embodiment 238, wherein the removing of unboundcontents from the plurality of scaffolds comprises washing the pluralityof scaffolds. Embodiment 248: The method of Embodiment 247, whereinwashing the plurality of scaffolds comprises rinsing the plurality ofscaffolds with a wash buffer. Embodiment 249: The method of Embodiment248, wherein the wash buffer is an aqueous buffer, an organic solution,or a mixture thereof. Embodiment 250: The method of Embodiment 247,wherein the plurality of scaffolds are subject to multiple wash andcollection steps, wherein each wash step comprises rinsing the pluralityof scaffolds with a wash buffer, and each collection step comprisescentrifugation or filtration of the plurality of scaffolds. Embodiment251: The method of Embodiment 238, wherein the liquid medium is anaqueous solution. Embodiment 252: The method of Embodiment 238, whereinthe liquid medium comprises an organic solvent. Embodiment 253: Themethod of Embodiment 238, wherein each scaffold of the plurality ofscaffolds is a bead, a fiber, a nanofibrous scaffold, a molecular cage,a dendrimer, or a multi-valent molecular assembly. Embodiment 254: Themethod of Embodiment 238, wherein each scaffold of the plurality ofscaffolds is polymer-bead, a glass bead, a metal bead, or a magneticbead. Embodiment 255: The method of Embodiment 238, wherein the liquidmedium comprises an amplification mix. Embodiment 256: The method ofEmbodiment 238, wherein each new encapsulation is a droplet. Embodiment257: The method of Embodiment 238, wherein encapsulating the pluralityof scaffolds in new encapsulations comprises introducing the pluralityof scaffolds into an emulsion. Embodiment 258: The method of Embodiment257, wherein introducing the plurality of scaffolds into an emulsioncomprises placing the plurality of scaffolds through a microfluidicdevice. Embodiment 259: The method of Embodiment 258, wherein themicrofluidic device is a microfluidic chip. Embodiment 260: The methodof Embodiment 257, wherein introducing the plurality of scaffolds intoan emulsion comprises placing the plurality of scaffolds into a one-potemulsifier. Embodiment 261: The method of Embodiments 238, wherein anamplification mix is encapsulated with the plurality of scaffolds in theplurality of new encapsulations. Embodiment 262: The method ofEmbodiment 238, wherein an amplification mix is added to the pluralityof new encapsulations. Embodiment 263: The method of Embodiment 262,wherein the amplification mix is added by pico-injection. Embodiment264: The method of embodiment 262, wherein the amplification mix isadded by droplet merging, wherein each encapsulation is a droplet.Embodiment 265: The method of Embodiment 261 or 262, wherein theamplification mix comprises PCR reagents. Embodiment 266: The method ofEmbodiment 238, further comprising sequencing the amplified nucleic acidencodings of each scaffold. Embodiment 267: The method of Embodiment238, wherein the method results in a lower background signal than anucleic acid encoded library that has not been subjected to the method.Embodiment 268: The method of Embodiment 267, wherein the backgroundsignal is reduced by at least 10%, at least 20%, at least 30%, at least40%, or at least 50%. Embodiment 269: The method of Embodiment 267,wherein the lower background signal allows for detection of nucleic acidencoded effectors whose encoding concentrations before the screen are100×, 1000×, 10000×, 100000×, or 1000000× lower in concentration thanthe average encoding concentration of the provided screened encodedeffectors and corresponding scaffolds.

Embodiment 270: A system for screening an encoded effector, the systemcomprising: a) a sample; b) a scaffold, wherein an encoded effector isbound to the scaffold by a cleavable linker, wherein a nucleic acidencoding the effector is bound to the scaffold; and c) a microfluidicdevice configured to: i) receive the sample and scaffold; ii)encapsulate the sample and scaffold within an encapsulation; iii) cleavethe cleavable linker from the encoded effector to release apredetermined amount of the encoded effector within the encapsulation;iv) incubate the encoded effector with the sample for an incubationperiod of time; v) detect a signal from the encapsulation, wherein thesignal results from an interaction between the encoded effector and thesample; and vi) sort the encapsulation based on the detection of thesignal. Embodiment 271: The system of Embodiment 270, wherein thecleavable linker is a photocleavable linker. Embodiment 272: The systemof Embodiment 271, wherein cleaving the photocleavable linker comprisesexposing the droplet to a light from a light source. Embodiment 273: Thesystem of Embodiment 272, wherein the light is UV light. Embodiment 274:The system of Embodiment 272, wherein the light intensity of the lightis from about 0.01 J/cm² to about 200 J/cm². Embodiment 275: The systemof Embodiment 271, wherein the encapsulation further encapsulates areagent configured to activate the photocleavable linker so as to enablethe photocleavable linker to be cleaved from the encoded effector.Embodiment 276: The system of Embodiment 275, wherein the microfluidicdevice is configured to introduce the reagent within the encapsulation.Embodiment 277: The system of Embodiment 270, wherein the signal isdetected based on detecting morphological changes in the sample measuredby recording a series of images of the droplet or detecting fluorescenceemitted by a molecular beacon or probe. Embodiment 278: The system ofEmbodiment 270, wherein the interaction between the encoded effector andthe cell comprises inhibition of one or more cellular components.Embodiment 279: The system of Embodiment 270, further comprising asequencing apparatus configured to identify the encoded effector bysequencing the nucleic acid encoding the effector. Embodiment 280: Thesystem of Embodiment 270, wherein the scaffold is a bead, a fiber, ananofibrous scaffold, a molecular cage, a dendrimer, or a multi-valentmolecular assembly. Embodiment 281: The system of Embodiment 280,wherein the scaffold is polymer-bead, a glass bead, a metal bead, or amagnetic bead. Embodiment 282: The system of Embodiment 280, wherein thebead is about 1 μm to about 100 μm in diameter. Embodiment 283: Thesystem of Embodiment 280, wherein the bead is about 1 μm to about 20 μmin diameter. Embodiment 284: The system of Embodiment 270, wherein theencoded effector is a peptide, a compound, protein, an enzyme, amacrocycle compound, or a nucleic acid. Embodiment 285: The system ofEmbodiment 284, wherein the encoded effector is a non-natural peptide ora polymer. Embodiment 286: The system of Embodiment 284, wherein theencoded effector is a small molecule or macromolecule. Embodiment 287:The system of Embodiment 284, wherein the compound is a drug-like smallmolecule. Embodiment 288: The system of Embodiment 270, wherein theencapsulation is a droplet. Embodiment 289: The system of Embodiment288, wherein the droplet is at most 1 picoliter, at most 10 picoliters,at most 100 picoliters, at most 1 nanoliter, at most 10 nanoliters, atmost 100 nanoliters, or at most 1 microliter in volume. Embodiment 290:The system of Embodiment 270, wherein the sample comprises at least onecell, a protein, or an enzyme. Embodiment 291: The system of Embodiment270, wherein the period of time is controlled by residence time as theencapsulation travels through a microfluidic channel of the microfluidicdevice. Embodiment 292: The system of Embodiment 270, wherein themicrofluidic device further comprises a first collection tube and secondcollection tube for sorting the encapsulation, wherein the encapsulationis placed in 1) the first collection tube if the signal is at or above apredetermined threshold or 2) the second collection tube if the signalis below a predetermined threshold. Embodiment 293: The system ofEmbodiment 292, further comprising a waveform pulse generator to movethe encapsulation to the first or second collection tube by anelectrical field gradient, by sound, by a diaphragm, by modifyinggeometry of the microfluidic channel, or by changing the pressure of amicrofluidic channel of the microfluidic device. Embodiment 294: Thesystem of Embodiment 270, wherein the microfluidic device furthercomprises: a) a first microfluidic channel comprising an aqueous fluidcomprising the sample and scaffold; b) a second microfluidic channelcomprising a fluid immiscible with the aqueous fluid; c) a junction atwhich the first microfluidic channel is in fluid communication with thesecond microfluidic channel, wherein the junction of the first andsecond microfluidic channels defines a device plane, wherein thejunction is configured to form the encapsulations of the aqueous fluidwithin the fluid from the second microfluidic channel, wherein the fluidfrom the second microfluidic channel with the encapsulations thereinmoves past the junction in a third microfluidic channel that defines anassay flow path; d) a cleavage region to cleave the cleavable linkerwithin the encapsulation disposed in the assay flow path; e) a detectionregion to detect the signal; and f) a sorting region to sort theencapsulation. Embodiment 295: The system of Embodiment 294, wherein thethird microfluidic channel is a continuation of the second microfluidicchannel. Embodiment 296: The system of Embodiment 294, wherein aplurality of encapsulations are disposed within the assay flow path.Embodiment 297: The system of Embodiment 294, wherein cleavage region isconfigured to expose each encapsulation to a light from a light sourceso as to cleave the encoded effector from the scaffold disposed withinthe assay flow path. Embodiment 298: The system of Embodiment 297,wherein the light intensity of the light is from about 0.01 J/cm² toabout 200 J/cm². Embodiment 299: The system of Embodiment 297, whereinthe plurality of encapsulations are exposed to a uniform intensity orduration of the light. Embodiment 300: The system of Embodiment 297,wherein the intensity or duration of the light that each encapsulationis exposed to within about 0.1% to about 10% of each other. Embodiment301: The system of Embodiment 270, wherein the incubation period of timefor each encapsulation is within a maximum dispersion ratio of theincubation period of time for the plurality of encapsulations, whereinthe dispersion ratio is based on a deviation about an average residencetime of the plurality of encapsulations passing through a region of themicrofluidic device. Embodiment 302: The system of Embodiment 301,wherein the region of the microfluidic device is the assay flow path.Embodiment 303: The system of Embodiment 301, wherein the maximumdispersion ratio is at most about 10%. Embodiment 304: The system ofEmbodiment 301, wherein the maximum dispersion ratio is at most about5%. Embodiment 305: The system of Embodiment 270, the incubation periodof time for each encapsulation is within about 0.1% to about 10% of eachother. Embodiment 306: The system of Embodiment 294, wherein thedetection region comprises a fluorometer. Embodiment 307: The system ofEmbodiment 294, wherein the detection region comprises a confocaldetection, laser scanning, or fluorescence, or combinations thereof.Embodiment 308: The system of Embodiment 294, wherein the sorting regioncomprises a sorter configured to sort the encapsulations based on asignal detected in the detection region.

Embodiment 309: A system for screening an encoded effector, the systemcomprising: a) one or more cells; b) a scaffold, wherein an encodedeffector is bound to the scaffold by a cleavable linker, wherein anucleic acid encoding the effector is bound to the scaffold; and c) amicrofluidic device configured to: i) receive the one or more cells andscaffold; ii) encapsulate the one or more cells and scaffold within anencapsulation; iii) cleave the cleavable linker from the encodedeffector to release a predetermined amount of the encoded effectorwithin the encapsulation; iv) incubate the encoded effector with the oneor more cells for an incubation period of time; v) detect a signal fromthe encapsulation, wherein the signal results from an interactionbetween the encoded effector and one or more cells; and vi) sort theencapsulation based on the detection of the signal. Embodiment 310: Thesystem of Embodiment 309, wherein the cleavable linker is aphotocleavable linker. Embodiment 311: The system of Embodiment 310,wherein cleaving the photocleavable linker comprises exposing thedroplet to a light from a light source. Embodiment 312: The system ofEmbodiment 311, wherein the light is UV light. Embodiment 313: Thesystem of Embodiment 311, wherein the light intensity of the light isfrom about 0.01 J/cm² to about 200 J/cm². Embodiment 314: The system ofEmbodiment 310, wherein the encapsulation further encapsulates a reagentconfigured to activate the photocleavable linker so as to enable thephotocleavable linker to be cleaved from the encoded effector.Embodiment 315: The system of Embodiment 314, wherein the microfluidicdevice is configured to introduce the reagent within the encapsulation.Embodiment 316: The system of Embodiment 309, wherein the signal isdetected based on detecting morphological changes in the one or morecells measured by recording a series of images of the droplet ordetecting fluorescence emitted by a molecular beacon or probe.Embodiment 317: The system of Embodiment 309, wherein the interactionbetween the encoded effector and the one or more cells comprisesinhibition of one or more cellular components. Embodiment 318: Thesystem of Embodiment 309, further comprising a sequencing apparatusconfigured to identify the encoded effector by sequencing the nucleicacid encoding the effector. Embodiment 319: The system of Embodiment309, wherein the scaffold is a bead, a fiber, a nanofibrous scaffold, amolecular cage, a dendrimer, or a multi-valent molecular assembly.Embodiment 320: The system of Embodiment 319, wherein the scaffold ispolymer-bead, a glass bead, a metal bead, or a magnetic bead. Embodiment321: The system of Embodiment 320, wherein the bead is about 1 μm toabout 100 μm in diameter. Embodiment 322: The system of Embodiment 320,wherein the bead is about 1 μm to about 20 μm in diameter. Embodiment323: The system of Embodiment 309, wherein the encoded effector is apeptide, a compound, protein, an enzyme, a macrocycle compound, or anucleic acid. Embodiment 324: The system of Embodiment 323, wherein theencoded effector is a non-natural peptide or a polymer. Embodiment 325:The system of Embodiment 323, wherein the encoded effector is a smallmolecule or macromolecule. Embodiment 326: The system of Embodiment 323,wherein the compound is a drug-like small molecule. Embodiment 327: Thesystem of Embodiment 309, wherein the encapsulation is a droplet.Embodiment 328: The system of Embodiment 327, wherein the droplet is atmost 1 picoliter, at most 10 picoliters, at most 100 picoliters, at most1 nanoliter, at most 10 nanoliters, at most 100 nanoliters, or at most 1microliter in volume. Embodiment 329: The system of Embodiment 309,wherein the period of time is controlled by residence time as theencapsulation travels through a microfluidic channel of the microfluidicdevice. Embodiment 330: The system of Embodiment 309, wherein themicrofluidic device further comprises a first collection tube and secondcollection tube for sorting the encapsulation, wherein the encapsulationis placed in 1) the first collection tube if the signal is at or above apredetermined threshold or 2) the second collection tube if the signalis below a predetermined threshold. Embodiment 331: The system ofEmbodiment 330, further comprising a waveform pulse generator to movethe encapsulation to the first or second collection tube by anelectrical field gradient, by sound, by a diaphragm, by modifyinggeometry of the microfluidic channel, or by changing the pressure of amicrofluidic channel of the microfluidic device. Embodiment 332: Thesystem of Embodiment 309, wherein the microfluidic device furthercomprises: a) a first microfluidic channel comprising an aqueous fluidcomprising the one or more cells and scaffold; b) a second microfluidicchannel comprising a fluid immiscible with the aqueous fluid; c) ajunction at which the first microfluidic channel is in fluidcommunication with the second microfluidic channel, wherein the junctionof the first and second microfluidic channels defines a device plane,wherein the junction is configured to form the encapsulations of theaqueous fluid within the fluid from the second microfluidic channel,wherein the fluid from the second microfluidic channel with theencapsulations therein moves past the junction in a third microfluidicchannel that defines an assay flow path; d) a cleavage region to cleavethe cleavable linker within the encapsulation disposed in the assay flowpath; e) a detection region to detect the signal; and f) a sortingregion to sort the encapsulation. Embodiment 333: The system ofEmbodiment 332, wherein the third microfluidic channel is a continuationof the second microfluidic channel. Embodiment 334: The system ofEmbodiment 332, wherein a plurality of encapsulations are disposedwithin the assay flow path. Embodiment 335: The system of Embodiment332, wherein cleavage region is configured to expose each encapsulationto a light from a light source so as to cleave the encoded effector fromthe scaffold disposed within the assay flow path. Embodiment 336: Thesystem of Embodiment 335, wherein the light intensity of the light isfrom about 0.01 J/cm² to about 200 J/cm². Embodiment 337: The system ofEmbodiment 335, wherein the plurality of encapsulations are exposed to auniform intensity or duration of the light. Embodiment 338: The systemof Embodiment 335, wherein the intensity or duration of the light thateach encapsulation is exposed to is within about 0.1% to about 10% ofeach other. Embodiment 339: The system of Embodiment 332, wherein theincubation period of time for each encapsulation is within a maximumdispersion ratio of the incubation period of time for the plurality ofencapsulations, wherein the dispersion ratio is based on a deviationabout an average residence time of the plurality of droplets passingthrough a region of the microfluidic device. Embodiment 340: The systemof Embodiment 339, wherein the region of the microfluidic device is theassay flow path. Embodiment 341: The system of Embodiment 339, whereinthe maximum dispersion ratio is at most about 10%. Embodiment 342: Thesystem of Embodiment 339, wherein the maximum dispersion ratio is atmost about 5%. Embodiment 343: The system of Embodiment 309, theincubation period of time for each encapsulation is within about 0.1% toabout 10% of each other. Embodiment 344: The system of Embodiment 332,wherein the detection region comprises a fluorometer. Embodiment 345:The system of Embodiment 332, wherein the detection region comprises aconfocal detection, laser scanning, or fluorescence, or combinationsthereof. Embodiment 346: The system of Embodiment 332, wherein thesorting region comprises a sorter configured to sort the encapsulationsbased on a signal detected in the detection region.

Embodiment 347: A microfluidic device for droplet based encoded libraryscreening comprising: a) a first microfluidic channel comprising anaqueous fluid; b) a second microfluidic channel comprising a fluidimmiscible with the aqueous fluid; c) a junction at which the firstmicrofluidic channel is in fluid communication with the secondmicrofluidic channel, wherein the junction of the first and secondmicrofluidic channels defines a device plane, wherein the junction isconfigured to form encapsulations of the aqueous fluid within the fluidfrom the second microfluidic channel, wherein the fluid from the secondmicrofluidic channel with the encapsulations therein moves past thejunction in a third microfluidic channel that defines an assay flowpath; d) a cleavage region for cleaving effectors bound to scaffoldsdisposed within the assay flow path; e) a detection region; and f) asorting region; g) wherein the device is configured for a dropletgeneration frequency of at least about 80 Hz. Embodiment 348: Themicrofluidic device of Embodiment 347, wherein the third microfluidicchannel is a continuation of the second microfluidic channel. Embodiment349: The microfluidic device of Embodiment 347, wherein the cleavageregion is upstream of the detection region and the sorting region.Embodiment 350: The microfluidic device of Embodiment 347, wherein thecleavage region is downstream of the junction. Embodiment 351: Themicrofluidic device of Embodiment 347, wherein the assay flow pathcomprises a serpentine flow path region. Embodiment 352: Themicrofluidic device of Embodiment 351, wherein the serpentine flow pathregion comprises at least 10, at least 20, at least 30, at least 40, atleast 50, or at least 100 curves. Embodiment 353: The microfluidicdevice of Embodiment 347, wherein the detection region comprises afluorometer. Embodiment 354: The microfluidic device of Embodiment 353,wherein the fluorometer is configured to have an optical axissubstantially parallel to the device plane. Embodiment 355: Themicrofluidic device of Embodiment 353, wherein the fluorometerilluminates a passing droplet at a curve in the assay flow path.Embodiment 356: The microfluidic device of Embodiment 353, wherein thefluorometer is configured to detect two or more wavelengths offluorescence. Embodiment 357: The microfluid device of Embodiment 347,wherein the detection region comprises a confocal detection, laserscanning, or fluorescence, or combinations thereof. Embodiment 358: Themicrofluidic device of Embodiment 347, wherein the device comprises twoor more channels comprising an aqueous fluid. Embodiment 359: Themicrofluidic device of Embodiment 347, wherein the detection region isupstream of the sorting region. Embodiment 360: The microfluidic deviceof Embodiment 347, wherein the sorting region comprises a sorterconfigured to sort droplets based on a signal detected in the detectionregion. Embodiment 361: The microfluidic device of Embodiment 347,wherein the assay flow path comprises one or more chambers disposedwithin the assay flow path. Embodiment 362: The microfluidic device ofEmbodiment 347, wherein the assay flow path comprises a plurality ofchambers disposed within the assay flow path, wherein the chambers areconnected by connecting channels. Embodiment 363: The microfluidicdevice of Embodiment 362, wherein the height of a chamber of theplurality of chambers is at most about 2× greater than the height of aconnecting channel of the plurality of connecting channels. Embodiment364: The microfluidic device of Embodiment 362, wherein the height ofthe chamber does not decrease until the width of the channel has beennarrowed to substantially match the width of the connecting channel.Embodiment 365: The microfluidic device of Embodiment 361, wherein theflow rate through the chambers is about 10% of the flow rate of the flowrate through the assay flow path upstream of the chambers. Embodiment366: The microfluidic device of Embodiment 347, wherein the device has adispersion ratio of at most about 10%. Embodiment 367: The microfluidicdevice of Embodiment 347, wherein the device is configured to incubatethe encapsulations for an incubation period of time, wherein theincubation period of time for each encapsulation is within a maximumdispersion ratio of the incubation period of time for the plurality ofencapsulations, wherein the dispersion ratio is based on a deviationabout an average residence time of the plurality of droplets passingthrough a region of the microfluidic device. Embodiment 368: The systemof Embodiment 367, wherein the region of the microfluidic device is theassay flow path. Embodiment 369: The system of Embodiment 368, whereinthe maximum dispersion ratio is at most about 10%. Embodiment 370: Thesystem of Embodiment 368, wherein the maximum dispersion ratio is atmost about 5%. Embodiment 371: The system of Embodiment 367, theincubation period of time for each encapsulation is within about 0.1% toabout 10% of each other.

Embodiment 372: A method for amplifying a primer to maximize cellularnucleic acid capture comprising: a) providing an encapsulationcomprising a nucleic acid encoded scaffold with one or more cells, anamplification mix, and a nicking enzyme, wherein a nucleic acid encodingis bound to the nucleic acid encoded scaffold; b) lysing the one or morecells to release one or more cellular nucleic acids; c) nicking thenucleic acid encoding with the nicking enzyme, thereby creating anencoded nucleic acid primer; d) amplifying the encoded nucleic acidprimer via the nicking site and amplification mix; and e) labeling areleased cellular nucleic acid with the encoded nucleic acid primer.Embodiment 373: The method of Embodiment 372, wherein the nicking enzymetargets a specific site in the nucleic acid encoding. Embodiment 374:The method of Embodiment 373, wherein the specific site comprises aspecific nucleotide sequence. Embodiment 375: The method of Embodimentof Embodiment 372, wherein amplifying the encoded nucleic acid primercomprises 1) creating a copy of the nucleic acid encoding that extendsfrom the nicking site, and 2) nicking the nucleic acid encoding copy tocreate another encoded nucleic acid primer. Embodiment 376: The methodof Embodiment of Embodiment 372, wherein amplifying the encoded nucleicacid primer comprises simultaneously 1) creating a copy of the nucleicacid encoding that extends from the nicking site, and 2) displacing thenucleic acid encoding copy to create another encoded nucleic acidprimer. Embodiment 377: The method of Embodiment 376, wherein theamplification mix comprises an amplification enzyme, such that theamplification enzyme enables for a copy of the nucleic acid encoding tobe simultaneously created and displaced. Embodiment 378: The method ofEmbodiment 377, wherein the amplification enzyme comprises a polymerase.Embodiment 379: The method of Embodiment 372, wherein each nucleic acidencoding comprises a capture site that prescribes a target cellularcoding or a target cellular nucleic acid to label a released cellularnucleic acid. Embodiment 380: The method of Embodiment 379, wherein thetarget nucleic acid is a target mRNA. Embodiment 381: The method ofEmbodiment 380, wherein the target mRNA encodes a protein of interest.Embodiment 382: The method of Embodiment 380, wherein the nicking enzymeenables an increase in target mRNA capture and labeling with the nucleicacid encoding. Embodiment 383: The method of Embodiment 380, whereintarget mRNA capture is increased by at least 10%, 25%, 50%, 100%, or200%. Embodiment 384: The method of Embodiment 372, wherein a pluralityof cellular nucleic acids are labeled with an respective encoded nucleicacid primer. Embodiment 385: The method of Embodiment 372, wherein thenucleic acid encoded scaffold comprises a bead, and the encoded nucleicacid primer comprises a unique bead barcode and an effector encoding.Embodiment 386: The method of Embodiment 372, wherein the encapsulationfurther comprises a cell lysis buffer. Embodiment 387: The method ofEmbodiment 372, wherein the encapsulation is a droplet, an emulsion, apicowell, a macrowell, a microwell, a bubble, or a microfluidicconfinement. Embodiment 388: The method of Embodiment 372, wherein theencapsulation is a droplet. Embodiment 389: The method of Embodiment388, wherein the droplet is at most 1 picoliter, at most 10 picoliters,at most 100 picoliters, at most 1 nanoliter, at most 10 nanoliters, atmost 100 nanoliters, or at most 1 microliter in volume. Embodiment 390:The method of Embodiment 372, wherein the amplification mix is anisothermal amplification mix. Embodiment 391: The method of Embodiment372, wherein the amplification mix comprises anicking-enzyme-amplification mixture. Embodiment 392: The method ofEmbodiment 372, wherein the amplification mix comprises a reversetranscriptase. Embodiment 393: The method of Embodiment 372, wherein thenucleic acid encoded scaffold is a bead, a fiber, a nanofibrousscaffold, a molecular cage, a dendrimer, or a multi-valent molecularassembly. Embodiment 394: The method of Embodiment 393, wherein thescaffold is polymer-bead, a glass bead, a metal bead, or a magneticbead. Embodiment 395: The method of Embodiment 372, wherein the nucleicacid encoded scaffold comprises an effector attached thereto. Embodiment396: The method of Embodiment 395, wherein the effector comprises acompound, a peptide, a protein, an enzyme, or a nucleic acid. Embodiment397: The method of Embodiment 395, wherein effector is attached to thescaffold by a cleavable linker. Embodiment 398: The method of Embodiment397, wherein the cleavable linker is cleaved by electromagneticradiation, an enzyme, chemical reagent, heat, pH adjustment, sound orelectrochemical reactivity. Embodiment 399: The method of Embodiment398, wherein the effector is cleaved from the scaffold usingelectromagnetic radiation. Embodiment 400: The method of Embodiment 398,wherein the amount of effector cleaved is controlled by the intensity orduration of exposure to electromagnetic radiation. Embodiment 401: Themethod of Embodiment 398, wherein the cleavable linker is cleaved usinga cleavage reagent. Embodiment 402: The method of Embodiment 401,wherein the amount of effector cleaved is controlled by theconcentration of the cleavage reagent in the encapsulation. Embodiment403: The method of Embodiment 398, wherein the rate of effector cleavageis controlled by the concentration of the cleavage reagent in theencapsulation. Embodiment 404: The method of Embodiment 398, wherein theeffector is cleaved from the scaffold using an enzyme. Embodiment 405:The method of Embodiment 404, wherein the enzyme is a protease, anuclease, or a hydrolase. Embodiment 406: The method of Embodiment 404,wherein the rate of effector cleavage is controlled by the amount ofenzyme in the encapsulation. Embodiment 407: The method of Embodiment372, wherein labeling a released cellular nucleic acids with the encodednucleic acid primer comprises barcoding the released cellular nucleicacid. Embodiment 408: The method of Embodiment 407, wherein theencapsulation further comprises barcoding reagents. Embodiment 409: Themethod of Embodiment 407, wherein barcoding the encoded nucleic acidprimer comprises adding barcoding reagents to the encapsulation.Embodiment 410: The method of Embodiment 408 or 409, wherein thebarcoding reagents comprise an enzyme or chemical cross-linking reagent.Embodiment 411: The method of Embodiment 410, wherein the barcodingreagents comprise an enzyme. Embodiment 412: The method of Embodiment411, wherein the enzyme is polymerase, a ligase, a restriction enzyme,or a recombinase. Embodiment 413: The method of Embodiment 410, whereinthe barcoding reagent is a chemical cross-linking reagent. Embodiment414: The method of Embodiment 413, wherein the chemical cross-linkingreagent is psoralen. Embodiment 415: The method of Embodiment 372,further comprising performing an effector screen, wherein the one ormore cells are being screened against an encoded effector. Embodiment416: The method of Embodiment 372, wherein the one or more cells areused to prepare the nucleic acid encoded scaffold for a screen.

Embodiment 417: A method for screening an encoded effector, the methodcomprising: a) providing an encapsulation comprising a sample and one ormore scaffolds, wherein the scaffold comprises: i) an encoded effectorbound to the scaffold by a cleavable linker and a nucleic acid encodingthe effector; b) adding one or more reagents to the encapsulationthrough pico-injection or by droplet merging; c) cleaving the cleavablelinker to release a pre-determined amount of the effector; d) detectingone or more signals from the encapsulation, wherein the signal resultsfrom an interaction between the encoded effector and the sample; and e)sorting the encapsulation based on the detection of the signal.Embodiment 418: The method of Embodiment 417, wherein the reagent isadded after a pre-determined amount of the effector has been released.Embodiment 419: The method of Embodiment 417, wherein the one or morereagents are added to the encapsulation by pico-injection. Embodiment420: The method of Embodiment 417, wherein the concentration a reagentof the one or more reagents is at most 100 picomolar (pM), at most 500pM, at most 1 nanomolar (nM), at most 10 nM, at most 100 nM, at most 1micromolar (04), at most 10 at most 100 at most 1 millimolar (mM), atmost 10 mM, at most 100 mM, or at most 500 mM. Embodiment 421: Themethod of Embodiment 417, wherein at least one reagent comprisesantibodies. Embodiment 422: The method of Embodiment 417, wherein thepredetermined amount of effector released from the scaffold is to aconcentration of at least 100 pM, at least 500 pM, at least 1 nM, atleast 10 nM, at least 100 nM, at least 1 at least 10 at least 100 atleast 1 mM. at least 10 mM, at least 50 mM, at least 100 mM, or at least250 mM. Embodiment 423: The method of Embodiment 417, wherein the samplecomprises at least one cell, a protein, an enzyme, a nucleic acid, acellular lysate, a tissue extract, or combinations thereof. Embodiment424: The method of Embodiment 417, at least one reagent comprises one ormore fluorophores. Embodiment 425: The method of Embodiment 417, furthercomprising barcoding the nucleic acid encoding the effector. Embodiment426: The method of Embodiment 425, wherein the barcoding is via the oneor more reagents added to the encapsulation. Embodiment 427: The methodof Embodiment 417, wherein the cleavable linker is a photocleavablelinker. Embodiment 428: The method of Embodiment 427, wherein thephotocleavable linker is cleaved using electromagnetic radiation.Embodiment 429: The method of Embodiment 427, wherein cleaving thephotocleavable linker comprises exposing the encapsulation to a lightfrom a light source. Embodiment 430: The method of Embodiment 429,wherein the light intensity of the light is from about 0.01 J/cm² toabout 200 J/cm². Embodiment 431: The method of Embodiment 427, whereinthe one or more reagents are configured to activate the photocleavablelinker, so as to enable the photocleavable linker to be cleaved from theencoded effector. Embodiment 432: The method of Embodiment 431, whereinat least one reagent is a disulfide reducing reagent. Embodiment 433:The method of Embodiment 431, wherein at least one reagent is atetrazine. Embodiment 434: The method of Embodiment 417, whereindetecting the signal comprises detecting morphological changes in thesample measured by recording a series of images of the droplet ordetecting fluorescence emitted by a molecular beacon or probe.Embodiment 435: The method of Embodiment 417, wherein the scaffold is abead, a fiber, a nanofibrous scaffold, a molecular cage, a dendrimer, ora multi-valent molecular assembly. Embodiment 436: The method ofEmbodiment 417, wherein the scaffold is polymer-bead, a glass bead, ametal bead, or a magnetic bead. Embodiment 437: The method of Embodiment435, wherein the bead is about 1 μm to about 100 μm in diameter.Embodiment 438: The method of Embodiment 435, wherein the bead is about1 μm to about 20 μm in diameter. Embodiment 439: The method ofEmbodiment 417, wherein the encoded effector is a peptide, a compound,protein, an enzyme, a macrocycle compound, or a nucleic acid. Embodiment440: The method of Embodiment 417, wherein the encapsulation is adroplet. Embodiment 441: The method of Embodiment 440, wherein thedroplet is at most 1 picoliter, at most 10 picoliters, at most 100picoliters, at most 1 nanoliter, at most 10 nanoliters, at most 100nanoliters, or at most 1 microliter in volume. Embodiment 442: Themethod of Embodiment 440, further comprising incubating the droplet fora period of time to allow the effector and the at least one cell tointeract. Embodiment 443: The method of Embodiment 417, wherein thesignal comprises electromagnetic radiation, thermal radiation, a visualchange in the sample, or combinations thereof.

Embodiment 444: A method for screening a library of encoded effectors,the method comprising: (a) encapsulating a plurality of beads into aplurality of droplets in a microfluidic channel with a sample, whereinthe plurality of beads are bound to a library of unique encodedeffectors, wherein each bead of the plurality of beads is bound to oneor more encoded effectors, wherein the library of unique encodedeffectors comprise at least about 250,000 unique effectors, wherein eachunique encoded effector is encoded with a unique nucleic acid encoding,wherein each droplet comprises one or more beads, (b) cleaving thephotocleavable linker between at least one encoded effector andcorresponding bead; (c) detecting a signal from one or more droplets ofthe plurality of droplets, wherein each signal results from aninteraction between a respective encoded effector and sample within thecorresponding droplet; and (d) sorting the plurality of droplets basedon the detection of a corresponding signal. Embodiment 445: The methodof Embodiment 444, wherein cleaving the photocleavable linker releases apredetermined amount of an encoded effector. Embodiment 446: The methodof Embodiment 445, wherein the predetermined amount of an encodedeffector released from the bead is to a concentration of at least 100pM, at least 500 pM, at least 1 nM, at least 10 nM, at least 100 nM, atleast 1 at least 10 at least 100 at least 1 mM. at least 10 mM, at least50 mM, at least 100 mM, or at least 250 mM. Embodiment 447: The methodof Embodiment 444, wherein the sample comprises at least one cell, aprotein, an enzyme, a nucleic acid, a cellular lysate, a tissue extract,or combinations thereof. Embodiment 448: The method of Embodiment 447,wherein the sample is one or more cells, a protein, or an enzyme.Embodiment 449: The method of Embodiment 447, further comprisingbarcoding a nucleic acid encoding a respective effector. Embodiment 450:The method of Embodiment 447, wherein the barcoding is via adding one ormore reagents to a droplet. Embodiment 451: The method of Embodiment447, wherein the photocleavable linker is cleaved using electromagneticradiation. Embodiment 452: The method of Embodiment 451, whereincleaving the photocleavable linker comprises exposing the encapsulationto a light from a light source. Embodiment 453: The method of Embodiment452, wherein the light intensity of the light is from about 0.01 J/cm²to about 200 J/cm². Embodiment 454: The method of Embodiment 447,wherein one or more reagents are added to a droplet, wherein the one ormore reagents are configured to activate the photocleavable linker of arespective encoded effector, so as to enable the photocleavable linkerto be cleaved from said encoded effector. Embodiment 455: The method ofEmbodiment 454, wherein the activating reagent is a disulfide reducingreagent. Embodiment 456: The method of Embodiment 454, wherein theactivating reagent is a tetrazine. Embodiment 457: The method ofEmbodiment 447, wherein detecting the signal comprises detectingmorphological changes in the sample measured by recording a series ofimages of the droplet or detecting fluorescence emitted by a molecularbeacon or probe. Embodiment 458: The method of Embodiment 447, whereinone or more beads is a polymer-bead, a glass bead, a metal bead, or amagnetic bead. Embodiment 459: The method of Embodiment 458, wherein oneor more beads is about 1 μm to about 100 μm in diameter. Embodiment 460:The method of Embodiment 458, wherein one or more beads is about 1 μm toabout 20 μm in diameter. Embodiment 461: The method of Embodiment 447,wherein an encoded effector is a peptide, a compound, protein, anenzyme, a macrocycle compound, or a nucleic acid. Embodiment 462: Themethod of Embodiment 447, wherein one or more droplets is at most 1picoliter, at most 10 picoliters, at most 100 picoliters, at most 1nanoliter, at most 10 nanoliters, at most 100 nanoliters, or at most 1microliter in volume. Embodiment 463: The method of Embodiment 447,further comprising incubating a droplet for a period of time to allowthe respective effector and the corresponding sample to interact.Embodiment 464: The method of Embodiment 447, wherein the signalcomprises electromagnetic radiation, thermal radiation, a visual changein the sample, or combinations thereof.

Disclosed herein, in some embodiments, is a method for screeningcombinations of encoded effectors against a sample, the methodcomprising: (a) amplifying a target protein within an encapsulation,wherein the encapsulation comprises: (i) a nucleic acid coding theexpression of the target protein, wherein the nucleic acid comprises abarcode region; and (ii) an in vitro transcription/translation system;(b) introducing two or more nucleic acid encoded effectors into theencapsulation, wherein the two or more nucleic acid encoded effectorscomprise nucleic acid encodings; (c) barcoding the nucleic acidencodings of the two or more encoded effectors using the barcode on thenucleic acid encoding the target protein; (d) incubating theencapsulation for a period of time to allow the two or more effectors tointeract with the target protein; and (e) measuring a signal produced bythe interaction between the two or more effectors and the targetprotein. In some embodiments, the method further comprising the step (f)sorting the encapsulation based on the measurement of the signal ascompared to a predetermined threshold. In some embodiments, the methodfurther comprising the step (g) sequencing the nucleic acid encoding theeffector which now comprises the barcode from the nucleic acid codingfor the target protein. In some embodiments, the method furthercomprising the step of (h) identifying combinations of effectors thatconferred efficacy against the target protein. In some embodiments,wherein amplifying the target protein comprises activating expression ofthe target protein. In some embodiments, wherein amplifying the targetprotein comprises expressing the protein to a desired concentration. Insome embodiments, the target protein is a signaling protein, an enzyme.,a binding protein, an antibody or antibody fragment, a structuralprotein, a storage protein, or a transport protein In some embodiments,the target protein is an enzyme. 99. In some embodiments, theencapsulation is a droplet. In some embodiments, the droplet is at most1 picoliter, at most 10 picoliters, at most 100 picoliters, at most 1nanoliter, at most 10 nanoliters, at most 100 nanoliters, or at most 1microliter in volume. In some embodiments, the barcoded nucleic acidencoding the target protein comprises a primer sequence complementary toa sequence on the one or more nucleic acids encoding the one or moreeffectors. In some embodiments, the barcoded nucleic acid coding theexpression of the target protein comprises a promoter sequence. In someembodiments, wherein introducing two or more nucleic acid encodedeffectors to the droplet comprises pico-injection or droplet merging. Insome embodiments, wherein two or more nucleic acid encoded effectors areintroduced into the encapsulation. In some embodiments, wherein at leasttwo nucleic acid encoded effectors are introduced into the droplet. Insome embodiments, wherein the nucleic acids encoding the two or moreeffectors are least 10, 15, 20, 25, 50, 75, or 100 nucleotides inlength. In some embodiments, wherein each nucleic acid encoding the oneor more effectors comprises a primer sequence complementary to asequence encoded on the barcoded nucleic acid coding the expression ofthe target protein. In some embodiments, wherein each effector is achemical compound. In some embodiments, wherein each effector is achemical fragment. In some embodiments, wherein at least one of thenucleic acids encoded effectors is attached to a scaffold. In someembodiments, the scaffold is a bead, a fiber, a nanofibrous scaffold, amolecular cage, a dendrimer, or a multi-valent molecular assembly. Insome embodiments, the scaffold is polymer-bead, a glass bead, a metalbead, or a magnetic bead. In some embodiments, the nucleic acid encodingthe effector is attached to the scaffold. In some embodiments, theattachment to the scaffold is through a cleavable linker. In someembodiments, the cleavable linker is cleavable by electromagneticradiation, an enzyme, chemical reagent, heat, pH adjustment, sound orelectrochemical reactivity. In some embodiments, the cleavable linker iscleavable by electromagnetic radiation. In some embodiments, the amountof effector, nucleic acid, or molecular weight barcode released can becontrolled by the intensity or duration of exposure to electromagneticradiation. In some embodiments, the cleavable linker is cleavable by acleaving reagent. In some embodiments, the cleavable linker is adisulfide bond or a substituted trans-cyclooctene, and the cleavingreagent is a phosphine or a tetrazine. In some embodiments, the amountof effector, nucleic acid, or molecular weight barcode released iscontrolled by the concentration of the chemical reagent in theencapsulation. In some embodiments, the rate of effector, nucleic acid,or molecular weight barcode released is controlled by the concentrationof the chemical reagent in the droplet. In some embodiments, thecleavable linker is cleavable by an enzyme. In some embodiments, theenzyme is a protease, a nuclease, or a hydrolase. In some embodiments,the rate of effector, nucleic acid, or molecular weight barcode releasedis controlled by the amount of enzyme in the droplet. In someembodiments, wherein barcoding the nucleic acids encoding the two ormore effectors with the barcode on the nucleic acid coding the targetprotein comprises hybridizing the one or more nucleic acids encoding theeffector with mRNA transcribed from the nucleic acid coding for thetarget protein and extending the transcribed mRNA or the nucleic acidencoding the effector with a polymerase enzyme. In some embodiments, theperiod of time is at least 1 minute, at least 10 minutes, at least 1hour, at least 4 hours, or at least 1 day. In some embodiments, theperiod of time is controlled by residence time as the droplet travelsthrough a microfluidic channel. In some embodiments, the residence timeis controlled by a flow rate through the microfluidic channel, ageometry of the microfluidic channel, a valve in the microfluidicchannel, or by removing the droplet from the microfluidic channel, ortransferring the droplet to a separate vessel. In some embodiments, thesignal is electromagnetic radiation, thermal radiation, or a visualchange in the sample. In some embodiments, the signal is electromagneticradiation. In some embodiments, the electromagnetic radiation is in thevisible spectrum. In some embodiments, the electromagnetic radiation isfluorescence or luminescence. In some embodiments, the signal isfluorescence emitted by a TaqMan probe or a molecular beacon. In someembodiments, the signal is thermal radiation detected with an infraredcamera. In some embodiments, the signal is a morphological of visualchange in the sample measured by recording a series of images of theencapsulation.

Disclosed herein, in some embodiments, is a method for screening anencoded effector without a physical sorting step, the method comprising:(a) providing a sample, a nucleic acid encoded effector, and a nucleicacid encoding in an encapsulation; (b) detecting a signal in theencapsulation resulting from an interaction between the effector and thesample; and (c) adding a first capping mix to the droplet based on thedetection, absence, or level of the signal, wherein the first cappingmix adds a first nucleic acid cap to the nucleic acid encoding. In someembodiments, the first nucleic acid cap comprises a first nucleic acidbarcode. In some embodiments, the first nucleic acid barcode indicatesthat the effector has a desired activity. In some embodiments, the firstnucleic acid cap is added to the nucleic acid encoding by ligation,hybridization, or extension of the nucleic acid encoding. In someembodiments, the first capping mix further comprises additional reagentsto effectuate the adding of the first nucleic acid cap. In someembodiments, the first nucleic acid cap is single-stranded DNA,double-stranded DNA, single-stranded RNA, or double-stranded RNA. Insome embodiments, the method further comprising the step of adding asecond capping mix to the encapsulation if the first capping mix is notadded to the encapsulation, wherein the second capping mix ads a secondnucleic acid cap to the nucleic acid encoding, wherein the first nucleicacid cap and the second nucleic acid cap have different sequences. Insome embodiments, the second nucleic acid cap comprises a second nucleicacid barcode. In some embodiments, the second nucleic acid barcodeindicates that the effector does not have a desired activity. In someembodiments, the second nucleic acid cap is added to the nucleic acidencoding by ligation, hybridization, or extension of the nucleic acidencoding. In some embodiments, the second capping mix further comprisesadditional reagents to effectuate the adding of the second nucleic acidcap. In some embodiments, the second nucleic acid cap is single-strandedDNA, double-stranded DNA, single-stranded RNA, or double-stranded RNA.In some embodiments, the second capping mix is added by pico-injection.In some embodiments, only the first capping mix or only the secondcapping mix is added to the encapsulation. In some embodiments, thefirst capping mix is added by pico-injection. In some embodiments, themethod does not comprise a further physical sorting of theencapsulations. In some embodiments, the sample is a biological sample.In some embodiments, the sample is one or more cells, one or moreproteins, one or more enzymes, one or more nucleic acids, one or morecellular lysates, or one or more tissue extracts. In some embodiments,the sample is a single cell. In some embodiments, the effector is acompound, a protein, a peptide, an enzyme, or a nucleic acid. In someembodiments, the effector is a compound. In some embodiments, theeffector is a drug-like small molecule. In some embodiments, the nucleicacid encoding comprises a terminal capping site. In some embodiments,the terminal capping site comprises a sequence complementary to asequence on the first nucleic acid cap. In some embodiments, the nucleicacid encoding comprises single-stranded DNA, double-stranded DNA,single-stranded RNA, or double-stranded RNA. In some embodiments, theencapsulation is a droplet. In some embodiments, the droplet is at most1 picoliter, at most 10 picoliters, at most 100 picoliters, at most 1nanoliter, at most 10 nanoliters, at most 100 nanoliters, or at most 1microliter in volume. In some embodiments, the encapsulation is anemulsion in an oil. In some embodiments, the effector is attached to ascaffold. In some embodiments, the scaffold is a bead, a fiber, ananofibrous scaffold, a molecular cage, a dendrimer, or a multi-valentmolecular assembly. In some embodiments, the scaffold is polymer-bead, aglass bead, a metal bead, or a magnetic bead. In some embodiments, theeffector is covalently attached to the scaffold by a first cleavablelinker. In some embodiments, the method further comprising cleaving thefirst cleavable linker. In some embodiments, the nucleic acid encodingis attached to the scaffold. In some embodiments, the nucleic acidencoding is covalently attached to the scaffold by a second cleavablelinker. In some embodiments, the first and second cleavable linkers aredifferent. In some embodiments, the method further comprising cleavingthe second cleavable linker. In some embodiments, the second cleavablelinker is cleaved prior to adding the first or second capping mix. Insome embodiments, the signal is electromagnetic radiation, thermalradiation, or a visual change in the sample. In some embodiments,detecting the signal comprises providing the encapsulation through amicrofluidic channel equipped with a detector. In some embodiments, themethod further comprising incubating the encapsulation for a period oftime to allow the effector and sample to interact. In some embodiments,the period of time is controlled by a residence time as theencapsulation travels through a microfluidic channel. In someembodiments, the method of further comprising sequencing the nucleicacid encoding. In some embodiments, the sequencing is next-generationsequencing. In some embodiments, the method comprising performing thescreen of any embodiment described herein against a library of encodedeffectors, wherein the library of encoded effectors comprises aplurality of different effectors.

In some embodiments, disclosed herein is a method of measuring effectorloading on scaffolds, the method comprising: (a) attaching an effectorsubunit to effector attachment sites on a plurality of scaffolds; (b)attaching a detectable label to any remaining free effector attachmentsites on the plurality of scaffolds after the step of attaching aneffector subunit; (c) removing a subset of scaffolds from the plurality;(d) measuring the amount of detectable label attached to the subset ofscaffolds to determine the amount of effector subunits successfullyattached to the effector attachment sites; (e) optionally activating theattached effector subunits to create new effector attachment sites; and(f) repeating steps (a)-(e) until a desired effector is assembled;wherein the scaffold further comprises a nucleic acid encoding theeffector or wherein the method further comprises attaching nucleic acidencoding subunits to the scaffold corresponding to the effector subunitsas the effector subunits are added to the scaffold. In some embodiments,In some embodiments, step (e) is omitted after the last effector subunitis attached. In some embodiments, each effector subunit attached to thescaffold is independently an amino acid, a small molecule fragment, anucleotide, or a compound. In some embodiments, each effector subunitattached to the scaffold is an amino acid. In some embodiments, eacheffector subunit attached to the scaffold is a compound. In someembodiments, the effector attachment sites comprise reactivefunctionalities. In some embodiments, the effector attachment sitescomprise amino or carboxylate groups. In some embodiments, the effectorattachment sites comprise biorthogonal or CLICK chemistry reactivegroups. In some embodiments, the effector subunits comprise a reactivegroup complementary to the effector attachment sites. In someembodiments, the detectable label comprises a reactive groupcomplementary to the effector attachment sites. In some embodiments, thedetectable label comprises a reactive group which is the same as areactive group on the effector subunit whose attachment is beingmeasured by the detectable label. In some embodiments, the detectablelabel is a fluorophore. In some embodiments, at most 10%, at most 20%,at most 30%, at most 40%, or at most 50% of the effector attachmentsites are free after the step of attaching the effector subunit. In someembodiments, removing a subset of the plurality of scaffolds comprisesremoving no more than 1%, no more than 2%, no more than 3%, no more than5%, or no more than 10% of the remaining scaffolds. In some embodiments,measuring the amount of detectable label attached to the subset ofscaffolds to determine the amount of effector subunits successfullyattached to the effector attachment sites comprises comparing themeasurement of the detectable label to the measurement of detectablelabel on a scaffold without any effector subunits attached. In someembodiments, the amount of effector subunits successfully attached tothe subset of scaffolds is expressed as a percentage of total attachmentsites occupied by the effector subunits. In some embodiments, optionallyactivating the attached effector subunits to create a new effectorattachment site comprises removing a protecting group from the attachedeffector subunit. In some embodiments, the protecting group is an aminoprotecting group, a carboxylate protecting group, an alcohol protectinggroup, a phenol protecting group, an alkyne protecting group, analdehyde protecting group, or a ketone protecting group. In someembodiments, the protecting group is an amino protecting group. In someembodiments, the amino protecting group is 9-fluorenylmethyloxcarbonyl(Fmoc), tert-butyloxycarbonyl (BOC), carbobenzyloxy (Cbz), benzyl (Bz),tosyl (Ts) or trichloroethyl chloroformate (Troc). In some embodiments,the protecting group is a carboxylate protecting group. In someembodiments, the carboxylate protecting group is a methyl ester, abenzyl ester, a tert-butyl ester, a 2,6-disubstituted phenolic ester, asilyl ester, or an orthoester. In some embodiments, the new effectorattachment site is the same functionality as the previous effectorattachment site. In some embodiments, the new effector attachment siteis a different functionality from the previous effector attachment site.In some embodiments, steps (a)-(e) are repeated at least 2, at least 3,at least 4, at least 5, at least 6, at least 7, at least 10, or at least20 times. 343. In some embodiments, the scaffold is a bead, a fiber, ananofibrous scaffold, a molecular cage, a dendrimer, or a multi-valentmolecular assembly. In some embodiments, the scaffold is polymer-bead, aglass bead, a metal bead, or a magnetic bead. In some embodiments, thescaffold comprises a nucleic acid encoding the effector. In someembodiments, the method further comprises attaching nucleic acidencoding subunits to the scaffold corresponding to the effector subunitsas the effector subunits are added to the scaffold. In some embodiments,a library of effector loaded scaffolds are synthesized concurrently. Insome embodiments, subsets of scaffolds from an effector attachment stepfrom the library are pooled prior to detection of the detectable labelIn some embodiments, the subsets of scaffolds are encapsulated in anencapsulation. In some embodiments, the encapsulations are droplets. Insome embodiments, a majority of the encapsulations comprise a singlescaffold. In some embodiments, scaffolds from the subset of scaffoldsare binned according to the amount of detectable label detected. In someembodiments, each bin comprises a unique range of detectable labeldetected. In some embodiments, for any method described herein, furthercomprising the step of sequencing encoding nucleic acids or encodingnucleic acid subunits of the pools to reveal which effector subunitscorrespond to a particular yield in a step of attaching effectorsubunits to effector attachment sites. In some embodiments, thesequencing step is performed each time steps (a)-(e) are repeated. Insome embodiments, yields of each step (a)-(e) for each unique scaffoldare collected to create a dataset which reveals the loading of thecomplete desired effector on each scaffold.

Disclosed herein, in some embodiments, is an array device for screeningencoded beads comprising: (a) a hydrophobic surface; and (b) nucleicacid patches interspersed on the hydrophobic surface; wherein thehydrophobic surface and nucleic acid patches are configured such thatwhen a proscribed amount of media is deployed across the surface eachnucleic acid patch is covered with media and the hydrophobic surfacebetween the nucleic acid patches does not contain media. In someembodiments, an array device described herein further comprising one ormore channels beneath the hydrophobic surface, wherein the channelscomprise terminal ends within nucleic acid patches. In some embodiments,the channels are configured to deliver reagents to the nucleic acidpatches. In some embodiments, the reagents are delivered as a liquidsolution. In some embodiments, the hydrophobic surface comprises ahydrophobic polymer. 3 In some embodiments, the hydrophobic polymercomprises a polyacrylic, a polyamide, a polycarbonate, a polydiene, apolyester, a polyether, a polyfluorocarbon, a polyolefin, a polystyrene,a polyvinyl acetal, a polyvinyl chloride, a polyvinyl ester, a polyvinylether, a polyvinyl ketone, a polyvinyl pyridine, a polyvinylpyrrolidone,a polysilane, a polyfluorosilane, a poly perfluorosilane or acombination thereof. In some embodiments, the hydrophobic polymercomprises a polyfluorocarbon. In some embodiments, the hydrophobicpolymer is fluorinated. In some embodiments, the hydrophobic surface isa surface functionalized with hydrophobic groups. In some embodiments,the hydrophobic groups are fatty acids, alkyl groups, alkoxy groups,aromatic groups, alkyl silanes, fluorosilanes, perfluorosilanes, orcombinations thereof. In some embodiments, the hydrophobic groups arefluorinated. In some embodiments, cells do not bind to the hydrophobicsurface. In some embodiments, the nucleic acid patches bind cells. Insome embodiments, single nucleic acid patches are encapsulated withinsingle droplets of the media. In some embodiments, the nucleic acidpatches comprise DNA, RNA, combinations thereof. In some embodiments,the nucleic acid patches comprise nucleic acids capable of bindingnucleic acid encoded beads. In some embodiments, the nucleic acids bindnucleic acid encoded beads non-specifically, by binding a complementarynucleic acid on the bead, or by binding another group on the bead. Insome embodiments, the nucleic acid patches are up to about 1 μm2 insize, up to about 10 μm2 in size, up to about 100 μm2 in size, up toabout 1000 μm2 in size, or up to about 10000 μm2 in size. In someembodiments, the nucleic acid patches are separated by up to about 1 μm,up to about 10 μm, up to about 100 μm, up to about 1000 μm, or up toabout 10000 μm. In some embodiments, the nucleic acid patches arearranged in a grid pattern. In some embodiments, the media is an aqueousmedia. In some embodiments, the density of nucleic acid patches is atleast 100 patches/cm2, at least 1000 patches/cm2, at least 10000patches/cm2, at least 100000 patches/cm2, at least 1000000 patches/cm2,or at least 10000000 patches/cm2. In some embodiments, the surface areaof the device is at least 1 cm2, at least 5 cm2, at least 10 cm2, atleast 25 cm2, at least 50 cm2, at least 100 cm2, at least 500 cm2, or atleast 1000 cm2.

Disclosed herein, in some embodiments, is a method of performing ascreen, the method comprising: (a) binding nucleic acid encoded beads tothe nucleic acid patches of the array of any one of embodimentsdescribed herein; (b) sequencing the nucleic acid encoded beads (c)binding cells to the nucleic acid patches; and (d) performing an assayon the array. In some embodiments, the beads further comprise encodedeffectors. In some embodiments, the method further comprising the stepof releasing the effectors from the beads. In some embodiments,releasing the effectors from the beads comprises adding a cleavingreagent to the nucleic acid patches. In some embodiments, sequencing thebeads allows determination of the physical location of specific nucleicacid encoded beads. In some embodiments, the assay produces a detectablesignal. 412. In some embodiments, each nucleic acid patch binds a singlebead and a single cell.

Disclosed herein, in some embodiments, is a method for stimulating anion channel, the method comprising: (a) providing a cell in anencapsulation; (b) stimulating an ion channel of the cell byelectrostimulation, optical stimulation, or chemical stimulation; and(c) detecting a signal from the cell by capturing images of the cell inthe encapsulation. In some embodiments, the ion channel is stimulated byelectrostimulation. In some embodiments, the electrostimulation isperformed by an electrode. In some embodiments, the electrode is withina flow path of the encapsulation. In some embodiments, the electrode isoutside of a flow path of the encapsulation. In some embodiments, theion channel is stimulated by optical stimulation. In some embodiments,the ion channel of the cell comprises a mutation. In some embodiments,the mutation sensitizes the ion channel to optical stimulation. In someembodiments, the ion channel is stimulated by chemical stimulation. Insome embodiments, the chemical stimulation comprises contacting the ionchannel with a toxin. In some embodiments, the toxin is added to theencapsulation by pico-injection. In some embodiments, the pico-injectionis conditional pico-injection. 4 In some embodiments, the toxin is anion channel toxin. In some embodiments, the signal is a morphological orvisual change in the cell. In some embodiments, capturing images of thecell comprises recording a series of images of the encapsulation. Insome embodiments, the method further comprising superimposing the seriesof images of the sample in the encapsulation. In some embodiments, theencapsulation further comprises a detection reagent.

In one aspect, provided herein, is a method for stimulating an ionchannel, the method comprising: (a) providing a cell in anencapsulation; (b) stimulating an ion channel of the cell byelectrostimulation, optical stimulation, or chemical stimulation; and(c) detecting a signal from the cell by capturing images of the cell inthe encapsulation.

In one aspect, provided herein, is a method for screening ion channelmodulators, the method comprising: (a) providing an encapsulationcomprising: (i) a cell expressing an ion channel protein; (ii) a set ofvoltage sensor probes; and (iii) an encoded effector and itscorresponding encoding; (b) stimulating an ion channel of the cell; and(c) detecting a signal from at least one member of the set of voltagesensor probes. In some embodiments, the encapsulation is a droplet, anemulsion, a picowell, a macrowell, a microwell, a bubble, or amicrofluidic confinement. In some embodiments, the encapsulation is adroplet. In some embodiments, the droplet is at most 1 picoliter, atmost 10 picoliters, at most 100 picoliters, at most 1 nanoliter, at most10 nanoliters, at most 100 nanoliters, or at most 1 microliter involume. In some embodiments, the cell comprises a mammalian cell. Insome embodiments, the cell comprises a human cell. In some embodiments,the cell comprises a HEK293 cell. In some embodiments, the ion channelprotein comprises a sodium, calcium, chloride, proton, or potassium ionchannel protein. In some embodiments, wherein the ion channel proteincomprises a voltage gated ion channel protein. In some embodiments, theion channel protein comprises an endogenous ion channel protein. In someembodiments, the ion channel protein comprises an exogenous ion channelprotein. In some embodiments, the ion channel protein comprises asodium, calcium, chloride, proton, or potassium voltage gated ionchannel protein. In some embodiments, the ion channel protein comprisesa voltage gated calcium channel protein (VGCC). In some embodiments, theion channel protein comprises an L-type calcium channel, a P-typecalcium channel, an N-type calcium channel, an R-type calcium channel,or a T-type calcium channel, or any mutant, fragment, or conjugatethereof. In some embodiments, the ion channel protein comprises achannelrhodopsin or any mutant, fragment, or conjugate thereof. In someembodiments, the channelrhodopsin is ChrimsonR or any mutant, fragment,or conjugate thereof. In some embodiments, the ion channel protein isoverexpressed. In some embodiments, the set of voltage sensor probescomprise a FRET pair. In some embodiments, the set of voltage sensorprobes comprises a voltage-sensitive oxonol, a fluorescent coumarin, orboth. In some embodiments, the set of voltage sensor probes comprises aDiSBAC compound, a coumarin phospholipid, or any combination orderivative thereof. In some embodiments, the set of voltage sensorscomprises a DiSBAC₂, DiSBAC₄, DiSBAC₆, CC1-DMPE, CC2-DMPE, or anycombination or derivative thereof. In some embodiments, the set ofvoltage sensors comprises DiSBAC₆ and CC2-DMPE. In some embodiments, theencapsulation further comprises a voltage assay background suppressioncompound. In some embodiments, the voltage assay background suppressioncompound comprises VABSC-1. In some embodiments, the effector and itscorresponding encoding are bound to a scaffold. In some embodiments, thescaffold is a bead, a fiber, a nanofibrous scaffold, a molecular cage, adendrimer, or a multi-valent molecular assembly. In some embodiments,the scaffold is polymer-bead, a glass bead, a metal bead, or a magneticbead. In some embodiments, the scaffold is a bead from 10 μm to about100 μm in diameter. In some embodiments, the effector is bound to thescaffold through a cleavable linker. In some embodiments, the cleavablelinker is a photocleavable linker. In some embodiments, the methodfurther comprises the step of cleaving the cleavable linker. In someembodiments, the effector is a compound or a peptide. In someembodiments, the effector is a small molecule. In some embodiments, theencoding is a nucleic acid. In some embodiments, stimulating the ionchannel comprises electrostimulation, optical stimulation, chemicalstimulation, or any combination thereof. In some embodiments,stimulating the ion channel comprises electrostimulation. In someembodiments, wherein stimulating the ion channel is performed by atleast one electrode. In some embodiments, the at least one electrode isin the flow path of the encapsulation. In some embodiments,electrostimulation is performed by non-contact electrodes to generateelectric fields, dielectrophoretic forces, or embedded metal-contactelectrodes. In some embodiments, electrostimulation is dictated bygeometry of a microfluidic device containing the encapsulation. In someembodiments, the frequency of electrostimulation is about 10 Hz. In someembodiments, stimulating the ion channel comprises optical stimulation.In some embodiments, the optical stimulation is UV, VIS, ornear-infrared radiation. In some embodiments, the optical stimulation isperformed using an embedded fiber-optic wave guide embedded in amicrofluidic device containing the encapsulation. In some embodiments,wherein the frequency of optical stimulation is about 10 Hz. In someembodiments, the wavelength of light for optical stimulation is about660 nm. In some embodiments, the intensity of light for opticalstimulation is about 500 mJ/s/cm². In some embodiments, stimulating theion channel comprises chemical stimulation. In some embodiments,chemical stimulation comprises contacting the ion channel with an ionchannel toxin. In some embodiments, the ion channel toxin comprisesveratridine, OD-1, or another ion channel toxin, or any combinationthereof. In some embodiments, the ion channel toxin as added to theencapsulation by pico-injection, droplet fusion, or through apre-arranged architecture of a microfluidic device which contains theencapsulation. In some embodiments, the signal is electromagneticradiation. In some embodiments, the electromagnetic radiation isluminescence or fluorescence. In some embodiments, the electromagneticradiation is fluorescence. In some embodiments, the electromagneticradiation is emitted due to a FRET interaction. In some embodiments, thesignal is an increase, decrease, or change in electromagnetic radiationas compared to an identical encapsulation without the encoded effector.In some embodiments, the signal is an increase, decrease, or change inelectromagnetic radiation as compared to the encapsulation before thestimulation of the ion channel. In some embodiments, the method furthercomprises the step of sorting the encapsulation based on the presence,absence, level, or change of the signal. In some embodiments, the methodfurther comprises measuring a property of the encoding to ascertain theidentity of the effector.

In one aspect, provided herein, is a microfluidic device for dropletbased encoded library screening comprising: (a) a first microfluidicchannel comprising an aqueous fluid; (b) a second microfluidic channelcomprising a fluid immiscible with the aqueous fluid; (c) a junction atwhich the first microfluidic channel is in fluid communication with thesecond microfluidic channel, wherein the junction of the first andsecond microfluidic channels defines a device plane, wherein thejunction is configured to form droplets of the aqueous fluid within thefluid from the second microfluidic channel, wherein the secondmicrofluidic channel is configured to continue past the junction therebydefining an assay flow path; (d) a cleavage region for cleavingeffectors from scaffolds disposed within the assay flow path; (e) adetection region; and (f) a sorting region. In some embodiments, thedevice further comprises a stimulation region. In some embodiments, thestimulation region comprises one or more actuators for stimulating anion channel. In some embodiments, the one or more actuators forstimulating the ion channel comprises at least one light source, atleast one electrode, or at least one pico-injection site equipped withan ion channel toxin. In some embodiments, the one or more actuatorscomprises at least one electrode. In some embodiments, the one or moreactuators comprises a pair of electrodes on opposite walls of the assayflow path such that when a droplet passes the pair of electrodes thedroplet contacts the electrodes, thereby allowing a current to flowthrough the droplet. In some embodiments, the stimulation regioncomprises at least 1, at least 2, at least 3, at least 5, at least 7, atleast 10, or at least 20 actuators. In some embodiments, at least one ofthe actuators for stimulating the ion channel is substantially parallelwith the device plane. In some embodiments, at least one of theactuators for stimulating the ion channel lies at a curve in the assayflow path. In some embodiments, the stimulation region is upstream ofthe detection region and downstream of the cleavage region. In someembodiments, the cleavage region comprises a light source configured tocleave effectors from scaffolds disposed within the assay flow path. Insome embodiments, the light source is a source of UV light. In someembodiments, the light source is configured to have an optical axissubstantially parallel with the device plane. In some embodiments, thelight source illuminates a passing droplet at a curve in the assay flowpath. In some embodiments, the cleavage region is upstream of thedetection region and the sorting region. In some embodiments, thecleavage region is downstream of the junction. In some embodiments, theassay flow path comprises a serpentine flow path region. In someembodiments, the serpentine flow path region comprises at least 10, atleast 20, at least 30, at least 40, at least 50, or at least 100 curves.In some embodiments, the detection region comprises a fluorometer. Insome embodiments, the fluorometer is configured to have an optical axissubstantially parallel to the device plane. In some embodiments, thefluorometer illuminates a passing droplet at a curve in the assay flowpath. In some embodiments, the fluorometer is configured to detect twoor more wavelengths of fluorescence. In some embodiments, the detectionregion is downstream of the cleavage region. In some embodiments, thedetection region is upstream of the sorting region. In some embodiments,the sorting region comprises a sorter configured to sort droplets basedon a signal detected in the detection region.

Definitions

Unless defined otherwise, all terms of art, notations and othertechnical and scientific terms or terminology used herein are intendedto have the same meaning as is commonly understood by one of ordinaryskill in the art to which the claimed subject matter pertains. In somecases, terms with commonly understood meanings are defined herein forclarity and/or for ready reference, and the inclusion of suchdefinitions herein should not necessarily be construed to represent asubstantial difference over what is generally understood in the art.

Throughout this application, various embodiments may be presented in arange format. It should be understood that the description in rangeformat is merely for convenience and brevity and should not be construedas an inflexible limitation on the scope of the disclosure. Accordingly,the description of a range should be considered to have specificallydisclosed all the possible subranges as well as individual numericalvalues within that range. For example, description of a range such asfrom 1 to 6 should be considered to have specifically disclosedsubranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4,from 2 to 6, from 3 to 6 etc., as well as individual numbers within thatrange, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of thebreadth of the range.

As used in the specification and claims, the singular forms “a”, “an”and “the” include plural references unless the context clearly dictatesotherwise. For example, the term “a sample” includes a plurality ofsamples, including mixtures thereof.

The terms “determining,” “measuring,” “detecting,” “evaluating,”“assessing,” “assaying,” and “analyzing” are often used interchangeablyherein to refer to forms of measurement. The terms include determiningif an element is present or not (for example, detection). These termscan include quantitative, qualitative or quantitative and qualitativedeterminations. Assessing can be relative or absolute. “Detecting thepresence of” can include determining the amount of something present inaddition to determining whether it is present or absent depending on thecontext.

The term “in vivo” is used to describe an event that takes place in asubject's body.

The term “ex vivo” is used to describe an event that takes place outsideof a subject's body. An ex vivo assay is not performed on a subject.Rather, it is performed upon a sample separate from a subject. Anexample of an ex vivo assay performed on a sample is an “in vitro”assay.

The term “in vitro” is used to describe an event that takes placescontained in a container for holding laboratory reagent such that it isseparated from the biological source from which the material isobtained. In vitro assays can encompass cell-based assays in whichliving or dead cells are employed. In vitro assays can also encompass acell-free assay in which no intact cells are employed.

The term “hit” refers to an effector that has been screened against asample and returned a positive result. The positive result may dependupon the nature of the screen being employed, but may include, withoutlimitation, an indication of efficacy against a target beinginterrogated.

The term “screen” as used herein refers to performing an assay using aplurality of effectors in order to determine the effect variouseffectors have on a particular sample.

The term “sequencing” refers to determining the nucleotide sequence of anucleic acid. Any suitable method for sequencing may be employed withthe methods and systems provided herein. The sequencing may beaccomplished by next generation sequencing. Next generation sequencingencompasses many kinds of sequencing such as pyrosequencing,sequencing-by-synthesis, single-molecule sequencing, second-generationsequencing, nanopore sequencing, sequencing by ligation, or sequencingby hybridization. Next-generation sequencing platforms are thosecommercially available from Illumina (RNA-Seq) and Helicos (Digital GeneExpression or “DGE”). Next generation sequencing methods include, butare not limited to those commercialized by: 1) 454/Roche Lifesciencesincluding but not limited to the methods and apparatus described inMargulies et al., Nature (2005) 437:376-380 (2005); and U.S. Pat. Nos.7,244,559; 7,335,762; 7,211,390; 7,244,567; 7,264,929; 7,323,305; 2)Helicos Biosciences Corporation (Cambridge, Mass.) as described in U.S.application Ser. No. 11/167,046, and U.S. Pat. Nos. 7,501,245;7,491,498; 7,276,720; and in U.S. Patent Application Publication Nos.US20090061439; US20080087826; US20060286566; US2006002471 1;US20060024678; US20080213770; and US20080103058; 3) Applied Biosystems(e.g. SOLiD sequencing); 4) Dover Systems (e.g., Polonator G.007sequencing); 5) lllumina, Inc. as described in U.S. Pat. Nos. 5,750,341;6,306,597; and 5,969,119; and 6) Pacific Biosciences as described inU.S. Pat. Nos. 7,462,452; 7,476,504; 7,405,281; 7,170,050; 7,462,468;7,476,503; 7,315,019; 7,302,146; 7,313,308; and US ApplicationPublication Nos. US20090029385; US20090068655; US20090024331; andUS20080206764. Such methods and apparatuses are provided here by way ofexample and are not intended to be limiting.

The term “barcode” refers to a nucleic acid sequence that is unique to aparticular system. The barcode may be unique to a particular method orto a particular effector. The nucleic acid encodings of the methods andsystems provided herein are analogous to barcodes in that they areunique nucleic acid sequences that can be used to identify the structureof a given effector. The length of a barcode or nucleic acid encodingshould be sufficient to differentiate between all the effectors in agiven library.

The term “flow” means any movement of liquid or solid through a deviceor in a method of the disclosure, and encompasses without limitation anyfluid stream, and any material moving with, within or against thestream, whether or not the material is carried by the stream. Forexample, the movement of molecules, cells or virions through a device orin a method of the disclosure, e.g. through channels of a microfluidicchip of the disclosure, comprises a flow. This is so, according to thedisclosure, whether or not the molecules, cells or virions are carriedby a stream of fluid also comprising a flow, or whether the molecules,cells or virions are caused to move by some other direct or indirectforce or motivation, and whether or not the nature of any motivatingforce is known or understood. The application of any force may be usedto provide a flow, including without limitation, pressure, capillaryaction, electro-osmosis, electrophoresis, dielectrophoresis, opticaltweezers, and combinations thereof, without regard for any particulartheory or mechanism of action, so long as molecules, cells or virionsare directed for detection, measurement or sorting according to thedisclosure.

An “inlet region” is an area of a microfabricated chip that receivesmolecules, cells or virions for detection measurement or sorting. Theinlet region may contain an inlet channel, a well or reservoir, anopening, and other features which facilitate the entry of molecules,cells or virions into the device. A chip may contain more than one inletregion if desired. The inlet region is in fluid communication with themain channel and is upstream therefrom.

An “outlet region” is an area of a microfabricated chip that collects ordispenses molecules, cells or virions after detection, measurement orsorting. An outlet region is downstream from a discrimination region,and may contain branch channels or outlet channels. A chip may containmore than one outlet region if desired.

An “analysis unit” is a microfabricated substrate, e.g., amicrofabricated chip, having at least one inlet region, at least onemain channel, at least one detection region and at least one outletregion. Sorting embodiments of the analysis unit include adiscrimination region and/or a branch point, e.g. downstream of thedetection region, that forms at least two branch channels and two outletregions. A device according to the disclosure may comprise a pluralityof analysis units.

A “main channel” is a channel of the chip of the disclosure whichpermits the flow of molecules, cells or virions past a detection regionfor detection (identification), measurement, or sorting. In a chipdesigned for sorting, the main channel also comprises a discriminationregion. The detection and discrimination regions can be placed orfabricated into the main channel. The main channel is typically in fluidcommunication with an inlet channel or inlet region, which permits theflow of molecules, cells or virions into the main channel. The mainchannel is also typically in fluid communication with an outlet regionand optionally with branch channels, each of which may have an outletchannel or waste channel. These channels permit the flow of cells out ofthe main channel.

A “detection region” is a location within the chip, typically within themain channel where molecules, cells or virions to be identified,measured or sorted on the basis of a predetermined characteristic. In anembodiment, molecules, cells or virions are examined one at a time, andthe characteristic is detected or measured optically, for example, bytesting for the presence or amount of a reporter. For example, thedetection region is in communication with one or more microscopes,diodes, light stimulating devices, (e.g., lasers), photo multipliertubes, and processors (e.g., computers and software), and combinationsthereof, which cooperate to detect a signal representative of acharacteristic, marker, or reporter, and to determine and direct themeasurement or the sorting action at the discrimination region. Insorting embodiments, the detection region is in fluid communication witha discrimination region and is at, proximate to, or upstream of thediscrimination region.

A “carrier fluid,” “immiscible fluid,” or “immiscible carrier fluid” orsimilar term as used herein refers to a liquid in which a sample orassay liquid is incapable of mixing and allows formation of droplets ofthe sample or assay liquid within the carrier fluid. These terms areused interchangeable herein and are meant to encompass the samematerials. Non-limiting examples of such carrier fluids include siliconbased oils, silicone oils, hydrophobic oils (e.g. squalene, fluorinatedoils, perfluorinated oils), or any fluid capable of encapsulatinganother desired liquid containing a sample to be analyzed.

An “extrusion region,” “droplet extrusion region,” or “droplet formationregion” is a junction between an inlet region and the main channel of achip of the disclosure, which permits the introduction of a pressurizedfluid to the main channel at an angle perpendicular to the flow of fluidin the main channel. In some embodiments, the fluid introduced to themain channel through the extrusion region is “incompatible” (i.e.,immiscible) with the fluid in the main channel so that droplets of thefluid introduced through the extrusion region are sheared off into thestream of fluid in the main channel.

A “discrimination region” or “branch point” is a junction of a channelwhere the flow of molecules, cells or virions can change direction toenter one or more other channels, e.g., a branch channel, depending on asignal received in connection with an examination in the detectionregion. Typically, a discrimination region is monitored and/or under thecontrol of a detection region, and therefore a discrimination region may“correspond” to such detection region. The discrimination region is incommunication with and is influenced by one or more sorting techniquesor flow control systems, e.g., electric, electro-osmotic, (micro-)valve, etc. A flow control system can employ a variety of sortingtechniques to change or direct the flow of molecules, cells or virionsinto a predetermined branch channel.

A “branch channel” is a channel which is in communication with adiscrimination region and a main channel. Typically, a branch channelreceives molecules, cells or virions depending on the molecule, cell orvirion characteristic of interest as detected by the detection regionand sorted at the discrimination region. A branch channel may be incommunication with other channels to permit additional sorting.Alternatively, a branch channel may also have an outlet region and/orterminate with a well or reservoir to allow collection or disposal ofthe molecules, cells or virions.

The term “forward sorting” or flow describes a one-direction flow ofmolecules, cells or virions, typically from an inlet region (upstream)to an outlet region (downstream), and in some instances without a changein direction, e.g., opposing the “forward” flow. In some embodiments,molecules, cells or virions travel forward in a linear fashion, i.e., insingle file. A “forward” sorting algorithm consists of runningmolecules, cells or virions from the input channel to the waste channel,until a molecule, cell or virion is identified to have an opticallydetectable signal (e.g. fluorescence) that is above a pre-set threshold,at which point voltages are temporarily changed to electro-osmoticallydivert the molecule or to the collection channel.

The term “reversible sorting” or flow describes a movement or flow thatcan change, i.e., reverse direction, for example, from a forwarddirection to an opposing backwards direction. Stated another way,reversible sorting permits a change in the direction of flow from adownstream to an upstream direction. This may be useful for moreaccurate sorting, for example, by allowing for confirmation of a sortingdecision, selection of particular branch channel, or to correct animproperly selected channel.

Different “sorting algorithms” for sorting in the microfluidic devicecan be implemented by different programs, for example under the controlof a personal computer. As an example, consider a pressure-switchedscheme instead of electro-osmotic flow. Electro-osmotic switching isvirtually instantaneous and throughput is limited by the highest voltagethat can be applied to the sorter (which also affects the run timethrough ion depletion effects). A pressure switched-scheme does notrequire high voltages and is more robust for longer runs. However,mechanical compliance in the system is likely to cause the fluidswitching speed to become rate-limiting with the “forward” sortingprogram. Since the fluid is at low Reynolds number and is completelyreversible, when trying to separate rare molecules, cells or virions,one can implement a sorting algorithm that is not limited by theintrinsic switching speed of the device. The molecules, cells or virionsflow at the highest possible static (non-switching) speed from the inputto the waste. When an interesting molecule, cell or virion is detected,the flow is stopped. By the time the flow stops, the molecule, cell orvirion may be past the junction and part way down the waste channel. Thesystem is then run backwards at a slow (switchable) speed from waste toinput, and the molecule, cell or virion is switched to the collectionchannel when it passes through the detection region. At that point, themolecule, cell or virion is “saved” and the device can be run at highspeed in the forward direction again. Similarly, a device of thedisclosure that is used for analysis, without sorting, can be run inreverse to re-read or verify the detection or analysis made for one ormore molecules, cells or virions in the detection region. This“reversible” analysis or sorting method is not possible with standardgel electrophoresis technologies (for molecules) nor with conventionalFACS machines (for cells). Reversible algorithms are particularly usefulfor collecting rare molecules, cells or virions or making multiple timecourse measurements of a molecule or single cell.

The term “emulsion” refers to a preparation of one liquid distributed insmall globules (also referred to herein as drops or droplets) in thebody of a second liquid. The first liquid, which is dispersed inglobules, is referred to as the discontinuous phase, whereas the secondliquid is referred to as the continuous phase or the dispersion medium.In one embodiment, the continuous phase is an aqueous solution and thediscontinuous phase is a hydrophobic fluid, such as an oil (e.g.,decane, tetradecane, or hexadecane). Such an emulsion is referred tohere as an oil in water emulsion. In another embodiment, an emulsion maybe a water in oil emulsion. In such an embodiment, the discontinuousphase is an aqueous solution and the continuous phase is a hydrophobicfluid such as an oil. The droplets or globules of oil in an oil in wateremulsion are also referred to herein as “micelles”, whereas globules ofwater in a water in oil emulsion may be referred to as “reversemicelles”.

As used herein, the term “about” a number refers to that number plus orminus 10% of that number. The term “about” a range refers to that rangeminus 10% of its lowest value and plus 10% of its greatest value.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

EXAMPLES

The following examples are included for illustrative purposes only andare not intended to limit the scope of the disclosure.

Example 1: Stoichiometric Cleavage of Encoded Effectors to ProbeProtease Inhibition

A library of beads containing nucleic acid encoded small molecules isprepared, wherein the small molecules are linked to the beads by asubstituted trans-cyclooctene. In this example, the library is beingscreened to detect small molecule inhibitors of trypsin. A solutioncomprising the library of beads is placed in a first reagent well 401 ofa microfluidic device 400, as shown in FIG. 4. A solution comprisingtrypsin is added to a reagent well 402, and an oil medium is added toreagent well 403. The contents of reagent wells 401, 402, and 403 flowuntil they meet at a junction 404, where the trypsin solution and beadsolution form droplets in an oil emulsion. The droplets then flow alongflow path 405 until they reach pico-injection site 406. Atpico-injection site 406, pico-injector 407 adds a solution containingdimethyl tetrazine and fluorescein isothiocyanate (FITC) labelledcasein. The pico-injection is configured such that each drop passing byreceives a uniform dose of the solution. The concentration of dimethyltetrazine in the solution is configured such that each dropletcomprising a bead releases substantially the same amount of effectorupon receiving the pico-injection. The droplet then continues along flowpath 405 until it reaches detector 408. Detector 408 is a fluorimeterconfigured to measure the FITC FRET emission (excitation 485 nm/emission538 nm). Based on the resulting fluorescence detected by detector 408,the sample is sorted at junction 409 onto a path toward bin 410 if theFRET emission is detected above a certain threshold and onto a pathtoward bin 411 if the FRET emission is not detected above the threshold.After the screen is completed, the nucleic acid encodings in bin 410 aresequenced by next generation sequencing to determine which smallmolecules had an inhibitory effect on trypsin.

Example 2: Nucleic Acid Detection with Molecular Beacons

A library of beads containing nucleic acid encoded small molecules isprepared, wherein the small molecules are linked to the beads by adisulfide bond. In this example, the library is being screened to detectan increase in cellular expression of a protein of interest by measuringcellular mRNA using molecular beacons. The molecular beacons used inthis example contains a sequence complementary to the mRNA which codesfor the protein of interest. The molecular beacons further comprises aCyanine 5 dye at one loop end and a DABCYL quencher at the other end. Inthis example, the protein of interest is expressed by a sample cell, andthe desired outcome of the screen is an increase in the expression ofthe protein of interest.

A solution comprising the library of beads is placed in a first reagentwell 501 of a microfluidic device 500, as shown in FIG. 5. A solutioncomprising the cells that express the protein of interest is added to areagent well 502, and an oil medium is added to reagent well 503. Thecontents of reagent wells 501, 502, and 503 flow until they meet at ajunction 504, where the solution containing the cells and the beadsolution form droplets in an oil emulsion. The device is configured suchthat a majority of the encapsulations receive a single cell and a singlebead. The droplets then flow along flow path 505 until they reachpico-injection site 506. At pico-injection site 506, pico-injector 507adds a solution containing tris(2-carboxyethyl)phosphine (TCEP). Thepico-injection is configured such that each drop passing by receives auniform dose of the solution. The concentration of TCEP in the solutionis configured such that each droplet comprising a bead releasessubstantially the same amount of effector upon receiving thepico-injection. The droplet then continues along flow path 505 until itreaches the second pico-injection site 508, at which point the molecularbeacon is added to the encapsulation, along with lysis buffer to lysethe cell, by pico-injector 509. The molecular beacons then hybridizewith any mRNA encoding the protein of interest, thereby allowing afluorescent emission from the Cyanine 5 moiety. The droplet continuesalong flow path 505 until it reaches the detector 510. Detector 510 is afluorimeter configured to measure the Cyanine 5 fluorescent signal(excitation 646 nm/emission 669 nm). Based on the resulting fluorescencedetected by detector 510, the sample is sorted at junction 511 onto apath toward bin 512 if the fluorescence emission is detected above acertain threshold and onto a path toward bin 513 if the fluorescenceemission is not detected above the threshold. After the screen iscompleted, the nucleic acid encodings in bin 512 are sequenced by nextgeneration sequencing to determine which small molecules had the desiredeffect of increasing production of the protein of interest.

Example 3: Screening of Mutant Imine Reductases

A library of beads containing nucleic acids coding for different mutantsof an imine reductase enzyme and a corresponding barcode is provided. Inthis example, the library is being screened to detect an enzyme that caneffectuate an imine reduction between Reagent 1

If the enzyme screened is capable of performing the imine reduction, theCyanine 3 and Cyanine 5 dyes will undergo a FRET interaction and anemission at 680 nm will be observed after an excitation at 540 nm.

A solution comprising the library of beads is placed in a first reagentwell 601 of a microfluidic device 600, as shown in FIG. 6. A solutioncomprising an in vitro transcription/translation system (IVTT) is thenadded to a reagent well 602, and an oil medium is added to reagent well603. The contents of reagent wells 601, 602, and 603 flow until theymeet at a junction 604, where the solution containing the IVTT and thebead solution form droplets in an oil emulsion. The device is configuredsuch that a majority of the encapsulations receive a single bead. TheIVTT then allows expression of the mutant imine reductases within thedroplets. The droplets then flow along flow path 605 until they reachpico-injection site 606. At pico-injection site 606, pico-injector 607adds a solution containing Reagent 1 and Reagent 2. The pico-injectionis configured such that each drop passing by receives a uniform dose ofthe solution. The droplet continues along flow path 605 until it reachesthe detector 508. Detector 510 is a fluorimeter configured to measurethe Cyanine 5/Cyanine 3 FRET emission (excitation 540 nm/emission 680nm). Based on the resulting fluorescence detected by detector 608, thesample is sorted at junction 609 onto a path toward bin 610 if thefluorescence emission is detected above a certain threshold and onto apath toward bin 611 if the fluorescence emission is not detected abovethe threshold. After the screen is completed, the nucleic acid on thebeads in bin 610 are sequenced by next generation sequencing todetermine which imine reductase mutants had the desired effect offorming the amine bond between Reagent 1 and Reagent 2 during thescreening

Example 4: Ion Channel Screening Using a Chip-Based Spatio-TemporallyControlled Electrical Stimulation Assay

A library of nucleic acid encoded beads containing candidate ion channelinhibitor molecules is prepared, wherein the inhibitor molecules arelinked to the beads by nitrobenzyl photocleavable linker. The cell lineused is the Human Embryonic Kidney (HEK) cell line that expresses asodium ion channel of interest. The cells are treated with a FRET probesystem, containing the dyes DiSBAC6 and CC2-DMPE which report a rapidchange in fluorescence upon the stimulation of an ion channel.Stimulation can occur by chemical, optical and electrical means.

In this example, electrical stimulation is used. The bead encodedlibrary is placed in reagent well 702 of microfluidic device 700, asshown in FIG. 7. The cell solution containing the FRET probe system isadded to reagent well 703, and an oil medium is added to reagent well701. From the reagent well 701, the oil travels along flow path 705. Thecontents of reagent wells 702 and 703 flow along separate flow pathsuntil they meet at junction 704. The aqueous sample solution flows downthe flow path channel until it meets the oil at junction 706, where thesolution containing the cells and the bead solution form an emulsionstream of aqueous droplets separated by the oil. The device can beconfigured such that a majority of the droplets form containing a singlecell and a single bead, but this is not necessary. The droplets thenflow along the flow path 705 until they reach UV light exposure site708, coupled to a UV source 707 by optical fiber, where the inhibitormolecule is released into the droplet from the nucleic acid encodedbead. As the droplet flows down the flow path 705, the candidateinhibitor contacts the cell. The droplet continues along the flow path705, where multiple electrodes 709 are placed along the flow path. Thedroplets are individually exposed to electrical stimulation at each setof electrodes 709. The electrode spacing and flow velocity defines thedesired stimulation frequency, which in this case is case 10 Hz. Afterstimulation, the droplets are passed through a fluorescence detectionregion 711, coupled to a light source and detector 710 by optical fiber.Droplets which contain effective inhibitors will exhibit a distinctlydifferent fluorescence intensity, after electrical stimulation, relativeto droplets that contain ineffective inhibitors. Droplets are thensorted at the sorting site 712 according to their distinctivefluorescence signal and are directed to collection bin 713 if designatedas a hit. Misses are directed to collection bin 714.

Example 5: Development of a Chip Device for Screening Ion ChannelModulators Phase 1 Goals:

Phase 1: To determine the feasibility of deploying anultra-high-throughput microfluidic system, Ion Channel Chip (IC Chip),to accommodate cell-based sodium ion channel activity assays. Proposethree different droplet microfluidic approaches to trigger cell surfaceion channel activities in a microfluidic chip by spatio-temporallycontrolled electrical-stimulation (ES); controlled optical-stimulation(OS), or by toxin-induced stimulation (TS) subsequent to compoundliberation and brief incubation. These methods will be tested todemonstrate sodium ion channel assay compatibility and screeningfeasibility in droplets. The aforementioned three methods of triggeringlive cell sodium ion channels may be executed. FIG. 8 shows an overviewof the development workflow for the design and evaluation of the devicesto accomplish the aforementioned methods.

The goal of Phase 1 is to demonstrate a proof-of-concept system usingknown inhibitor control compounds photolytically released from carrierbeads in droplets. The released compound will inhibit Na+ ion channelactivity in cells with sufficient discretion when compared touninhibited cells from a model cell line.

Objectives:

Stage 1: Detection of Droplet-Cell-Assay for Ion Channel Inhibition

Summary: Demonstrate cell line compatibility, and cell assay monitoringin microdroplets via DiSBAC₆+CC2-DMPE FRET probe emission. This earlyproof-of-concept will rely on merging cell suspension with stimulatorytoxin (Veratridine, etc.) just prior to droplet-formation, followed byemission detection. The goal of this stage is to optimize the opticalinterrogation techniques and quantitate the confidence of discerning aninhibited ion channel cell population from a control population, withindroplets in flow. Measuring fluorogenic probe emission in system is abasic requirement for further development of in-droplet stimulationmethods within Stage 2. In addition, will initiate cloning and selectionof a channel rhodopsin expressing cell line, unless is able to provide acell line capable of optogenetic stimulation that is compatible with thecapabilities of for detection, to evaluate optical stimulation methodsin electrophysiology well-plates and control assays.

Milestone:

Satisfy a benchmark of ≥10% ratio amplitude (+/−inhibitor) after toxinstimulation at peak or during the tail-current, whichever is moresensitive. A Z′-score calculation can also be applied to determinestatistical confidence in separating inhibited from control populationsif ratio amplitude is not relevant for toxin stimulation.

Stage 2: Stimulation of Droplet-Cell-Assay with Spatio-Temporal Controland Design and Construction of a 10K Member “Targeted Library” AgainstDesired Ion Channel

Summary:

Demonstrate the ability to stimulate cells in droplets, in flow, by oneor more of three methods (ES, OS, or TS) using appropriately designed ICchips (see FIG. 1). Subsequent to stimulation, demonstrate detection ofDiSBAC6+CC2-DMPE FRET emission at an optimal time-point to segregateinhibited cell population from a positive control. In addition, thedesign of a 10K member “targeted library” around chemotypes present inthe control molecules used in Stage 3 will be demonstrated usingchemoinformatic tools to maximize the structural and chemical diversityof the 10K member targeted library. The initial synthetic methodologiesused to construct the library will be tested and the chemical productsof the methodologies will be validated with LC/MS analysis. Lastly, the“targeted library” will be subjected to UV-cleavage to demonstrate therelease of library members from BELs and the degree of cleavage will bequantified with LC/MS analysis.

Milestone:

Stimulation method and timing must satisfy a qualifying Z′≥0.4beten+/−inhibitor cell populations. This is a basic requirement forfurther microfluidic development to include compound delivery, dosing,and sorting of inhibited cells within Stage 3. The “targeted library”will be constructed with yields >30% for each individual library memberusing the desired synthetic methodologies and the library shoulddemonstrate the ability to be cleaved from beads using UV-cleavagemethods

Stage 3: Complete Integrated Chip Design for POC Screen Using Controlsand a Subsequent Screen of a 10K Member Targeted Library

Summary:

Integrate the cell-in-droplet stimulation architectures into a completeintegrated device, including in-situ release of control inhibitors,pre-stimulation mixing and incubation, and post-stimulation sorting.Upon validation of a full integrated chip and validation of controlmolecules for inhibiting stimulation, a “targeted library” will bescreened against a desired ion channel to elucidate SAR around thecontrols from the which the “targeted library” is derived. The “targetedlibrary” will be screened across 5 concentrations. Analytical tools willbe created to automate NGS analysis, decode “active” structures, rankactive members by potency, and provide insights into SAR. A validationworkflow will be established, to resynthesize the most potent candidatemolecules for analysis and profiling using's standard assays to verifyEC₅₀.

Milestone:

Positive control inhibitor beads will be sampled, and a dose-responsemeasured across 6 concentrations. Data will be used to determine arelative EC₅₀ within system, which must be within 3× of the known EC₅₀.Sorting will also be demonstrated, capable of isolating positive controlbeads from negative controls with <10% false-sort events. The outputfrom a “targeted library” screen will up to 10 visualizations of the rawdata output from the screen.

Methodology and Project Design: Definitions

Probe=DiSBAC₆ or analog, with CC2-DMPE unless otherwise statedToxin=Veratridine, OD-1 or other stimulatory moleculeModel cell line=HEK293 (Human Embryonic Kidney cells) unless otherwisestated.Inhibitor=provided control compounds for system testing.ChR=Channel rhodopsin or variant with tuned optical properties.Fluorescent nuclear stains=DAPI, DRAQ5, PicoGreen, etc.IC chip=Ion Channel chip to initiate stimulation of sodium ion channelsin droplets.ICES=Ion Channel chip designed for electrical stimulation ofcells-in-droplet.ICTS=Ion Channel chip designed for toxin stimulation ofcells-in-droplet.ICOS=Ion Channel chip designed for optogenetic stimulation ofcells-in-droplet

Stage 1: Detection of Droplet-Cell-Assay for Ion Channel Inhibition

1A) A model cell-line expressing a relevant ion channel will be used anda simple set of basic controls established to verify all reagents and tounderstand the dynamic activity of toxin stimulation by probe emission.

-   -   1) well-plate control assay using model cell line, with probe,        +/−inhibitor control using toxin stimulation.    -   2) Fluorescence microscopy will determine cell-line uniformity,        and steady-state behavior for probe emission, +/−inhibitor.    -   3) FRET emission profiles of bulk population in plates collected        to understand toxin kinetics, steady-state, and EC₅₀,        +/−inhibitor.

1B) A simple microfluidic droplet generator will be used to introducemodel cells with fluor-labels (Nuclear stain, fluor-Anti-ion channel) orprobe to test cell detection in droplets in flow.

-   -   1) Fluor-Anti-ion channel or similar label will be ideal to        determine cell-expression uniformity, and to tune optical        detection in flow using photo-stable fluorophores to determine        the optical sensitivity for the system, without the variable of        probe leeching or photo-bleaching.    -   2) Membrane bound probe emission (CC2-DMPE) detection in droplet        will then finalize the sensitivity required to observe probe        emission in droplet in a flow channel.    -   3) Model cell line biocompatibility and toxicity measurements        inside droplets using fluorogenic live or dead stains.

1C) Develop IC_(TS) Chip v1.0) to merge cells+probe (+/−inhibitor), withstimulatory toxin, just prior to droplet formation then capture probeemission from cells at set time points

-   -   1) Flow-velocity in addition to the spatial position of        excitation and detection dictates the time-delay post        stimulation for assay observation.    -   2) Toxin stimulation generates a depolarization pulse followed        by a persistent tail current. Detection position can isolate        specific points on this curve and can determine the best        location for differentiating+/−inhibitor cell populations.    -   3) Probe emission profiles for cells+/−inhibitor will be        compared, and a statistical confidence (Z′-score) determined at        various time points following toxin stimulation.

1D) Channelrhodopsin expression cell line generation to prove outoptogenetic stimulation

-   -   1) Ion channel expression in HEK cell line for electrical stim        (or sced from)    -   2) Ion Channel+ChR (ChrimsonR or other variant, DOI:        10.1038/nmeth.2836) expression in model cell line for optical        stimulation.

1E) Cell stimulation control in electrophysiology microplates,+/−inhibitor.

-   -   1) Electrode stimulation in well-plate using ion channel        expression cell line, observing emission from fluorogenic probe        (DiSBAC6+CC2-DMPE) as a control for cell line quality.    -   2) Optical stimulation in electrode well-plate using ion        channel+ChR to detect current.        -   A) Require >99% spike occurrence from ChrimsonR stimulation            at 10 Hz using 660-nm light at 500 mJ/s/cm².    -   3) Optical stimulation in well-plate using ion channel+ChR+probe        to detect probe emission response.

Stage 2: Stimulation of Droplet-Cell-Assay with Spatio-Temporal Controland Design and Construction of a 10K Member “Targeted Library” AgainstIon Channel

2A) Covalent, photo-cleavable attachment of control inhibitors to beads(positive control bead.

-   -   1) to collaborate and provide control inhibitors to contain        reactive handles for attachment to photo-cleavable linker.        -   a) Ideally suited are free primary or secondary amine,            carboxylic acid, terminal amide, or phenol.    -   2) Full product cleavage and LC-MS to verify        photolabile-compound linkage.    -   3) Photocleavage of inhibitor in well-plate to verify activity        after UV release.

2B) Design and fabrication of IC chips for cell-in-droplet stimulation,monitoring probe emission by PMT or imaging. This stage is a significantengineering effort with multiple strategies to prove out the most viablecandidates for Stage 3.

-   -   1) IC_(ES)—Electrical stimulation in droplet will be examined        using two approaches, with frequency of stimulation dictated by        geometry (10 Hz).        -   A) Non-contact electrodes to generate electric fields or            dielectrophoretic forces.    -   2) IC_(TS)—Toxin stimulation in droplet will be examined using a        pico-injection, droplet fusion, or a pre-injected architecture,        which allows for stimulatory toxin to be injected into droplet        post compound dosing in Stage 3.    -   3) IC_(OS)—Optical stimulation in droplet will be examined using        an embedded fiber-optic waveguide illuminating cells with either        UV, VIS or NIR wavelengths at geometry defined frequencies (10        Hz).

2C) IC chip demonstrations showing clear differentiation between cellpopulations+/−inhibitor. See FIG. 8 for strategy overview.

-   -   1) IC_(ES) and IC_(OS) chips will be designed for 10 Hz        stimulation pulses, and probe-emission monitored at spatially        controlled time-delays post stimulation to evaluate the assay        Z′-score between +/−inhibitor cell populations at different        time-points.        -   A) Inhibitor titration (5-point) and detection will create            an end-point dose-response profile to compare the            approximate potency to ‘s standard assays.    -   2) IC_(TS) chip design will be tested using the time-interval        post stimulatory toxin-injection determined in Stage 1C-2 to        evaluate the assay Z’-score between +/−inhibitor cell        populations in dose response to compare potency to's standard        assays.        -   A) Inhibitor titration (5-point) and detection will create            an end-point dose-response profile to compare the            approximate potency to's standard assays.

2D) Design of 10K member “targeted library” and validation of syntheticmethodologies used to construct the library.

-   -   1) The design of the library will utilize chemistries, as        desired to permute the chemical structure of control compounds        of known activity. The library will be designed so as to        maximize the interpolation of structure-activity-relationships        (SAR) of individual library members.    -   2) The synthetic methodologies used to construct the library        will be validated with “building blocks” representative of        “building block” classes used to construct the library. The        yields of reactions with individual building blocks will be        quantified with LC/MS to validate the reactivity of individual        building blocks.    -   3) Individual beads from the “targeted library” will be        subjected to photo-cleavage to verify that library members are        cleaved from beads in the library.

Stage 3: Complete Integrated Chip Design for POC Screen Using Controlsand a Subsequent Screen of a 10K Member “Targeted Library

3A) Candidate IC chip designs with qualifying Z′-scores will beincorporated into a complete integrated system.

-   -   1) IC_(xx) chip 2.0 designed, fabricated, and tuned for        inhibitor-bead delivery, compound dosing, incubation,        cell-in-droplet stimulation, assay detection, and droplet        sorting.    -   2) POC of IC chip 2.0 devices using positive control beads,        releasing high-concentrations of inhibitor to optimize Z′-score        within the integrated system.    -   3) Demonstration of inhibitor-bead isolation from negative-bead        control by droplet sorting with <10% false-sort events (droplets        not containing inhibitor-bead and cells.

3B) Compound-release trio-calibration curve to enable predictivecompound dosing.

-   -   1) Fluorophore concentration vs PMT detection calibration in        droplet.    -   2) Fluorophore release from bead by UV exposure vs PMT detection        calibration in droplet.    -   3) UV exposure vs calibrant dye emission calibration.

3C) Control-bead titration with cell-population analysis to showcasedose-response and approximation of EC₅₀ for control inhibitor

-   -   1) Bead-released inhibitor titration and assay detection across        4 Ln (i.e. 10 μM, 3 μM, 300 nM, <100 nM).    -   2) The inferred IC₅₀ of bead released compound in IC chip needs        to be <3× from that shown using standard plate methods with the        same model cell line.

3D)) “Targeted Library” screen against ion channel using the designedlibrary from Stage 2.

-   -   1) The “targeted” library will be screened, using 7 library        equivalents, against ion channel with “spiked-in” positive        control compounds on beads used in 3C.    -   2) Data will be analyzed with chemoinformatic tools and will be        presented at the conclusion of the screen within 1 month of the        screen being performed.

While preferred embodiments of the present disclosure have been shownand described herein, it will be obvious to those skilled in the artthat such embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the disclosure. It should beunderstood that various alternatives to the embodiments of thedisclosure described herein may be employed in practicing thedisclosure. It is intended that the following claims define the scope ofthe disclosure and that methods and structures within the scope of theseclaims and their equivalents be covered thereby.

1.-20. (canceled)
 21. A system comprising: (a) a plurality ofcompartments, wherein each compartment of a subset of the plurality ofcompartments comprises: (i) a cell; and (ii) a scaffold comprising: (A)an effector bound to the scaffold via a cleavable linker, wherein theeffector is releasable from the scaffold via cleavage of the cleavablelinker to generate a released effector into the compartment; and (B) afirst barcode corresponding to and for identifying the effector; and (b)at least one of: (i) a sorting device for sorting at least one of: acompartment, contents of a compartment, a scaffold of a compartment, afirst barcode of a compartment, or a cell of a compartment, of thesubset of the plurality of compartments, based on a property of a signalindicative of an effect of the released effector on the cell; (ii) atagging reagent present in the subset of the plurality of compartmentscomprising a second barcode for tagging the scaffold or the effectorbased on a property of a signal indicative of an effect of the releasedeffector on the cell; or (iii) an injection module for adding aninjected reagent into a compartment of the subset of the plurality ofcompartments based on a property of a signal indicative of an effect ofthe released effector on the cell.
 22. The system of claim 21, whereinthe compartment is a droplet, a well, a micro-well, a microfluidicconfinement, or a combination thereof.
 23. The system of claim 21,wherein the scaffold is a particle with a diameter of at least about 1micrometer (μm).
 24. The system of claim 21, wherein the effectorcomprises a subunit of the effector, and the first barcode comprises asubunit of the first barcode corresponding to and for identifying theeffector or the subunit of the effector.
 25. The system of claim 21,wherein the cleavable linker is a photocleavable linker configured torelease the effector upon exposure to light.
 26. The system of claim 25,further comprising a light source for exposing the cleavable linker tolight to release the effector from the scaffold.
 27. The system of claim21, wherein the first barcode is a nucleic acid molecule,deoxyribonucleic acid (DNA), ribonucleic acid (RNA), a peptide, or apeptide nucleic acid (PNA).
 28. The system of claim 21, wherein thesubset of the plurality of compartments further comprises an assayreagent for assessing an effect of the released effector on the cell andproducing a signal indicative of the effect.
 29. The system of claim 28,wherein the assay reagent comprises a probe for generating the signalupon interacting with the cell.
 30. The system of claim 21, wherein thesorting device performs the sorting via generating a stimulus forstimulating or initiating a movement of the compartment, the contents ofthe compartment, the scaffold, the barcode, the cell, or any combinationthereof, based on the signal.
 31. The system of claim 30, wherein thestimulus comprises a pulse, a voltage differential or charge gradient,an electrical field gradient, a sound, a pressure differential orgradient, a radiation pressure, an optical stimulus, a light beam orlaser, electro-osmosis, electrophoresis, dielectrophoresis, mechanicalswitching, gravity, a change in the geometry of a microfluidic channelor channel squeezing, fluid flow, or any combination thereof.
 32. Thesystem of claim 21, wherein the system further comprises a waveformpulse generator, an electrode, an electrical field generator, an opticaltweezer, a light source or laser, a diaphragm, a pressure source, a flowreservoir, tubing and tubing connection, a valve, a pump, a pressurecontroller, electro-osmotic flow control, an electrophoresis module, adielectrophoresis module, a collection chamber or container, a celltrapper, an injection module, a computer, an algorithm, or anycombination thereof.
 33. The system of claim 21, further comprising adetector for detecting the signal.
 34. The system of claim 33, whereinthe detector comprises a computer, an optical detector, a microscope, acamera, an image processing device, a light source, a laser, a photoncounter, a computer, a microprocessor, an algorithm, a computer program,or any combination thereof.
 35. The system of claim 21, wherein theinjection module comprises a pico-injection module or a droplet mergingmodule.
 36. The system of claim 21, wherein the tagging comprisesattaching the first barcode to the second barcode.
 37. The system ofclaim 36, wherein the first barcode and the second barcode are nucleicacid molecules, and wherein the attaching comprises ligating the secondbarcode to the first barcode, annealing the second barcode to the firstbarcode, hybridizing the second barcode to the first barcode, amplifyingthe second barcode onto the first barcode, extending the first barcode,or any combination thereof.
 38. The system of claim 21, wherein thefirst barcode comprises a first sequence, the second barcode comprises asecond sequence, and the first sequence is complementary to the secondsequence.
 39. The system of claim 21, wherein the first barcode furtheridentifies the scaffold.
 40. The system of claim 39, wherein the firstbarcode is a nucleic acid barcode comprising an effector-specificsequence for identifying the effector, and a scaffold-specific sequencefor identifying the scaffold.
 41. The system of claim 21, wherein theinjected reagent comprises a cleaving reagent for cleaving the firstbarcode or the effector, a lysis buffer for lysing the cell, an assayreagent, the second barcode, or any combination thereof.